pSK41/pGO1-family conjugative plasmids of Staphylococcus aureus encode a cryptic repressor of replication.

The majority of large multiresistance plasmids of Staphylococcus aureus utilise a RepA_N-type replication initiation protein, the expression of which is regulated by a small antisense RNA (RNAI) that overlaps the rep mRNA leader. The pSK41/pGO1-family of conjugative plasmids additionally possess a small (86 codon) divergently transcribed ORF (orf86) located upstream of the rep locus. The product of pSK41 orf86 was predicted to have a helix-turn-helix motif suggestive of a likely function in transcriptional repression. In this study, we investigated the effect of Orf86 on transcription of thirteen pSK41 backbone promoters. We found that Orf86 only repressed transcription from the rep promoter, and hence now redesignate the product as Cop. Over-expression of Cop in trans reduced the copy number of pSK41 mini-replicons, both in the presence and absence of rnaI. in vitro protein-DNA binding experiments with purified 6 × His-Cop demonstrated specific DNA binding, adjacent to, and partially overlapping the -35 hexamer of the rep promoter. The crystal structure of Cop revealed a dimeric structure similar to other known transcriptional regulators. Cop mRNA was found to result from "read-through" transcription from the strong RNAI promoter that escapes the rnaI terminator. Thus, PrnaI is responsible for transcription of two distinct negative regulators of plasmid copy number; the antisense RNAI that primarily represses Rep translation, and Cop protein that can repress rep transcription. Deletion of cop in a native plasmid did not appear to impact copy number, indicating a cryptic auxiliary role.

The Plasmidomic Landscape of Clinical Methicillin-Resistant Staphylococcus aureus Isolates from Malaysia.

Methicillin-resistant Staphylococcus aureus (MRSA) is a priority nosocomial pathogen with plasmids playing a crucial role in its genetic adaptability, particularly in the acquisition and spread of antimicrobial resistance. In this study, the genome sequences of 79 MSRA clinical isolates from Terengganu, Malaysia, (obtained between 2016 and 2020) along with an additional 15 Malaysian MRSA genomes from GenBank were analyzed for their plasmid content. The majority (90%, 85/94) of the Malaysian MRSA isolates harbored 1-4 plasmids each. In total, 189 plasmid sequences were identified ranging in size from 2.3 kb to ca. 58 kb, spanning all seven distinctive plasmid replication initiator (replicase) types. Resistance genes (either to antimicrobials, heavy metals, and/or biocides) were found in 74% (140/189) of these plasmids. Small plasmids (<5 kb) were predominant (63.5%, 120/189) with a RepL replicase plasmid harboring the ermC gene that confers resistance to macrolides, lincosamides, and streptogramin B (MLSB) identified in 63 MRSA isolates. A low carriage of conjugative plasmids was observed (n = 2), but the majority (64.5%, 122/189) of the non-conjugative plasmids have mobilizable potential. The results obtained enabled us to gain a rare view of the plasmidomic landscape of Malaysian MRSA isolates and reinforces their importance in the evolution of this pathogen.

Complete Genome Sequence and Analysis of a ST573 Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus SauR3 Clinical Isolate from Terengganu, Malaysia.

Methicillin-resistant Staphylococcus aureus (MRSA) is a World Health Organization-listed priority pathogen. Scarce genomic data are available for MRSA isolates from Malaysia. Here, we present the complete genome sequence of a multidrug-resistant MRSA strain SauR3, isolated from the blood of a 6-year-old patient hospitalized in Terengganu, Malaysia, in 2016. S. aureus SauR3 was resistant to five antimicrobial classes comprising nine antibiotics. The genome was sequenced on the Illumina and Oxford Nanopore platforms and hybrid assembly was performed to obtain its complete genome sequence. The SauR3 genome consists of a circular chromosome of 2,800,017 bp and three plasmids designated pSauR3-1 (42,928 bp), pSauR3-2 (3011 bp), and pSauR3-3 (2473 bp). SauR3 belongs to sequence type 573 (ST573), a rarely reported sequence type of the staphylococcal clonal complex 1 (CC1) lineage, and harbors a variant of the staphylococcal cassette chromosome mec (SCCmec) type V (5C2&5) element which also contains the aac(6')-aph(2″) aminoglycoside-resistance genes. pSauR3-1 harbors several antibiotic resistance genes in a 14,095 bp genomic island (GI), previously reported in the chromosome of other staphylococci. pSauR3-2 is cryptic, whereas pSauR3-3 encodes the ermC gene that mediates inducible resistance to macrolide-lincosamide-streptogramin B (iMLSB). The SauR3 genome can potentially be used as a reference genome for other ST573 isolates.

Comparative Genomic Analysis of a Multidrug-Resistant Staphylococcus hominis ShoR14 Clinical Isolate from Terengganu, Malaysia, Led to the Discovery of Novel Mobile Genetic Elements.

Staphylococcus hominis is a coagulase-negative Staphylococcus (CoNS) commensal capable of causing serious systemic infections in humans. The emergence of multidrug-resistant S. hominis strains is of concern but little is known about the characteristics of this organism, particularly from Malaysia. Here, we present the comparative genome analysis of S. hominis ShoR14, a multidrug-resistant, methicillin-resistant blood isolate from Terengganu, Malaysia. Genomic DNA of S. hominis ShoR14 was sequenced on the Illumina platform and assembled using Unicycler v0.4.8. ShoR14 belonged to sequence type (ST) 1 which is the most prevalent ST of the S. hominis subsp. hominis. Comparative genomic analysis with closely related strains in the database with complete genome sequences, led to the discovery of a novel variant of the staphylococcal chromosome cassette mec (SCCmec) type VIII element harboring the mecA methicillin-resistance gene in ShoR14 and its possible carriage of a SCCfus element that encodes the fusidic acid resistance gene (fusC). Up to seven possible ShoR14 plasmid contigs were identified, three of which harbored resistance genes for tetracycline (tetK), chloramphenicol (catA7), macrolides, lincosamides, and streptogramin B (ermC). Additionally, we report the discovery of a novel mercury-resistant transposon, Tn7456, other genomic islands, and prophages which make up the S. hominis mobilome.

Evolving origin-of-transfer sequences on staphylococcal conjugative and mobilizable plasmids-who's mimicking whom?

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.

Evolution of a 72-Kilobase Cointegrant, Conjugative Multiresistance Plasmid in Community-Associated Methicillin-Resistant Staphylococcus aureus Isolates from the Early 1990s.

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 ß-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.

Staphylococcal Plasmids, Transposable and Integrative Elements.

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.

Construction of Challenging Proline-Proline Junctions via Diselenide-Selenoester Ligation Chemistry.

Polyproline sequences are highly abundant in prokaryotic and eukaryotic proteins, where they serve as key components of secondary structure. To date, construction of the proline-proline motif has not been possible owing to steric congestion at the ligation junction, together with an n → π* electronic interaction that reduces the reactivity of acylated proline residues at the C-terminus of peptides. Here, we harness the enhanced reactivity of prolyl selenoesters and a trans-γ-selenoproline moiety to access the elusive proline-proline junction for the first time through a diselenide-selenoester ligation-deselenization manifold. The efficient nature of this chemistry is highlighted in the high-yielding one-pot assembly of two proline-rich polypeptide targets, submaxillary gland androgen regulated protein 3B and lumbricin-1. This method provides access to the most challenging of ligation junctions, thus enabling the construction of previously intractable peptide and protein targets of increasing structural complexity.

Mobile Genetic Elements Associated with Antimicrobial Resistance.

Strains of bacteria resistant to antibiotics, particularly those that are multiresistant, are an increasing major health care problem around the world. It is now abundantly clear that both Gram-negative and Gram-positive bacteria are able to meet the evolutionary challenge of combating antimicrobial chemotherapy, often by acquiring preexisting resistance determinants from the bacterial gene pool. This is achieved through the concerted activities of mobile genetic elements able to move within or between DNA molecules, which include insertion sequences, transposons, and gene cassettes/integrons, and those that are able to transfer between bacterial cells, such as plasmids and integrative conjugative elements. Together these elements play a central role in facilitating horizontal genetic exchange and therefore promote the acquisition and spread of resistance genes. This review aims to outline the characteristics of the major types of mobile genetic elements involved in acquisition and spread of antibiotic resistance in both Gram-negative and Gram-positive bacteria, focusing on the so-called ESKAPEE group of organisms (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli), which have become the most problematic hospital pathogens.

Replication of Staphylococcal Resistance Plasmids.

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.

An updated view of plasmid conjugation and mobilization in Staphylococcus.

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.

Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci.

UNLABELLED: 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 multiresistance plasmid pSK41 by its relaxase, NES. This enzyme also processed variant oriT-like sequences found on numerous plasmids previously considered nontransmissible, suggesting that in conjunction with an uncharacterized accessory protein, these plasmids may be transferred horizontally via a relaxase in trans mechanism. These findings have important implications for our understanding of staphylococcal resistance plasmid evolution.

Structural and sequence requirements for the antisense RNA regulating replication of staphylococcal multiresistance plasmid pSK41.

pSK41 is a prototypical 46-kb conjugative multiresistance plasmid of Staphylococcus aureus. The pSK41 replication initiation protein (Rep) is rate-limiting for plasmid replication, and its expression is negatively regulated by a small, non-coding antisense transcript, RNAI, that is complementary to the rep mRNA leader region. In this study, enzymatic probing was used to verify the predicted secondary structures of RNAI and its target RNA. We demonstrated that two stem-loop structures of RNAI, SLRNAI-II and SLRNAI-III, were important for inhibition. A putative U-turn motif detected in the loop of SLrep-I (5'-UUGG-3') was analysed for its significance to RNAI-mediated inhibition in vivo and Northern blotting suggested that rep mRNA was processed. Taken together, these observations support our previously proposed model but also raise new questions about the replication control mechanism.

Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein.

The staphylococcal multiresistance plasmids are key contributors to the alarming rise in bacterial multidrug resistance. A conserved replication initiator, RepA, encoded on these plasmids is essential for their propagation. RepA proteins consist of flexibly linked N-terminal (NTD) and C-terminal (CTD) domains. Despite their essential role in replication, the molecular basis for RepA function is unknown. Here we describe a complete structural and functional dissection of RepA proteins. Unexpectedly, both the RepA NTD and CTD show similarity to the corresponding domains of the bacterial primosome protein, DnaD. Although the RepA and DnaD NTD both contain winged helix-turn-helices, the DnaD NTD self-assembles into large scaffolds whereas the tetrameric RepA NTD binds DNA iterons using a newly described DNA binding mode. Strikingly, structural and atomic force microscopy data reveal that the NTD tetramer mediates DNA bridging, suggesting a molecular mechanism for origin handcuffing. Finally, data show that the RepA CTD interacts with the host DnaG primase, which binds the replicative helicase. Thus, these combined data reveal the molecular mechanism by which RepA mediates the specific replicon assembly of staphylococcal multiresistant plasmids.

Staphylococcus aureus surface proteins involved in adaptation to oxacillin identified using a novel cell shaving approach.

Staphylococcus aureus is a Gram-positive pathogen responsible for a variety of infections, and some strains are resistant to virtually all classes of antibiotics. Cell shaving proteomics using a novel probability scoring algorithm to compare the surfaceomes of the methicillin-resistant, laboratory-adapted S. aureus COL strain with a COL strain in vitro adapted to high levels of oxacillin (APT). APT displayed altered cell morphology compared with COL and increased aggregation in biofilm assays. Increased resistance to ß-lactam antibiotics was observed, but adaptation to oxacillin did not confer multidrug resistance. Analysis of the S. aureus COL and APT surfaceomes identified 150 proteins at a threshold determined by the scoring algorithm. Proteins unique to APT included the LytR-CpsA-Psr (LCP) domain-containing MsrR and SACOL2302. Quantitative RT-PCR showed increased expression of sacol2302 in APT grown with oxacillin (>6-fold compared with COL). Overexpression of sacol2302 in COL to levels consistent with APT (+ oxacillin) did not influence biofilm formation or ß-lactam resistance. Proteomics using iTRAQ and LC-MS/MS identified 1323 proteins (∼50% of the theoretical S. aureus proteome), and cluster analysis demonstrated elevated APT abundances of LCP proteins, capsule and peptidoglycan biosynthesis proteins, and proteins involved in wall remodelling. Adaptation to oxacillin also induced urease proteins, which maintained culture pH compared to COL. These results show that S. aureus modifies surface architecture in response to antibiotic adaptation.

Biology of the staphylococcal conjugative multiresistance plasmid pSK41.

Plasmid pSK41 is a large, low-copy-number, conjugative plasmid from Staphylococcus aureus that is representative of a family of staphylococcal plasmids that confer multiple resistances to a wide range of antimicrobial agents. The plasmid consists of a conserved plasmid backbone containing the genes for plasmid housekeeping functions, which is punctuated by copies of IS257 that flank a Tn4001-hybrid structure and cointegrated plasmids that harbour resistance genes. This review summarises the current understanding of the biology of pSK41, focussing on the systems responsible for its replication, maintenance and transmission, and their regulation.

Molecular basis of antibiotic multiresistance transfer in Staphylococcus aureus.

Multidrug-resistant Staphylococcus aureus infections pose a significant threat to human health. Antibiotic resistance is most commonly propagated by conjugative plasmids like pLW1043, the first vancomycin-resistant S. aureus vector identified in humans. We present the molecular basis for resistance transmission by the nicking enzyme in S. aureus (NES), which is essential for conjugative transfer. NES initiates and terminates the transfer of plasmids that variously confer resistance to a range of drugs, including vancomycin, gentamicin, and mupirocin. The NES N-terminal relaxase-DNA complex crystal structure reveals unique protein-DNA contacts essential in vitro and for conjugation in S. aureus. Using this structural information, we designed a DNA minor groove-targeted polyamide that inhibits NES with low micromolar efficacy. The crystal structure of the 341-residue C-terminal region outlines a unique architecture; in vitro and cell-based studies further establish that it is essential for conjugation and regulates the activity of the N-terminal relaxase. This conclusion is supported by a small-angle X-ray scattering structure of a full-length, 665-residue NES-DNA complex. Together, these data reveal the structural basis for antibiotic multiresistance acquisition by S. aureus and suggest novel strategies for therapeutic intervention.

Genetic requirements for replication initiation of the staphylococcal multiresistance plasmid pSK41.

Replication of staphylococcal multiresistance plasmid pSK41 is initiated by binding of the replication initiator protein (Rep) to the Rep boxes, a series of four direct repeats located centrally within the rep gene. A Staphylococcus aureus strain was engineered to provide Rep in trans, allowing localization of the pSK41 origin of replication (oriV) to a 185 bp segment, which included the Rep boxes and a series of downstream direct repeats. Deletion analysis of individual Rep boxes revealed that all four Rep boxes are required for maximum origin activity, with the deletion of one or more Rep boxes having a significant effect on the proficiency of replication. However, a hierarchy of importance was identified among the Rep boxes, which appears to be mediated by the minor sequence variations that exist between them. DNA binding studies with truncated Rep proteins have enabled the DNA binding domain to be localized to the N-terminal 134 amino acids of the protein.

Analysis of the prototypical Staphylococcus aureus multiresistance plasmid pSK1.

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 28.15kb nucleotide sequence of pSK1 and discuss the genetic content and evolution of the 14kb 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.

Complete nucleotide sequence and comparative analysis of pPR9, a 41.7-kilobase conjugative staphylococcal multiresistance plasmid conferring high-level mupirocin resistance.

We have sequenced the conjugative plasmid pPR9, which carries the ileS2 gene, which had contributed to the dissemination of high-level mupirocin resistance at our institution. The plasmid backbone shows extensive genetic conservation with plasmids belonging to the pSK41/pGO1 family, but comparative analyses have revealed key differences that provide important insights into the evolution of these medically important plasmids and high-level mupirocin resistance in staphylococci and highlight the role of insertion sequence IS257 in these processes.