Finbarr Hayes

The University of Manchester, Manchester, England, United Kingdom

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Publications (60)330.13 Total impact

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    ABSTRACT: The ability to assemble DNA sequences de novo through efficient and powerful DNA fabrication methods is one of the foundational technologies of synthetic biology. Gene synthesis, in particular, has been considered the main driver for the emergence of this new scientific discipline. Here we describe RapGene, a rapid gene assembly technique which was successfully tested for the synthesis and cloning of both prokaryotic and eukaryotic genes through a ligation independent approach. The method developed in this study is a complete bacterial gene synthesis platform for the quick, accurate and cost effective fabrication and cloning of gene-length sequences that employ the widely used host Escherichia coli.
    Scientific Reports 06/2015; DOI:10.1038/srep11302 · 5.58 Impact Factor
  • Sadia Saeed · Thomas A Jowitt · Jim Warwicker · Finbarr Hayes ·
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    ABSTRACT: The ribbon-helix-helix (RHH) superfamily of DNA binding proteins is dispersed widely in procaryotes. The dimeric RHH fold is generated by interlocking of two monomers into a two-fold symmetrical structure that comprises four α-helices enwrapping a pair of antiparallel β-strands (ribbon). Residues in the ribbon region are the principal determinants of DNA binding, whereas the RHH hydrophobic core is assembled from amino acids in both the α-helices and ribbon element. The ParG protein encoded by multiresistance plasmid TP228 is a RHH protein that functions dually as a centromere binding factor during segrosome assembly and as a transcriptional repressor. Here we identify residues in the α-helices of ParG that are critical for DNA segregation and in organization of the protein hydrophobic core. A key hydrophobic aromatic amino acid at one position was functionally substitutable by other aromatic residues, but not by non-aromatic hydrophobic amino acids. Nevertheless, intramolecular suppression of the latter by complementary change of a residue that approaches nearby from the partner monomer fully restored activity in vivo and in vitro. The interactions involved in assembling the ParG core may be highly malleable and suggest that RHH proteins are tractable platforms for the rational design of diverse DNA binding factors useful for synthetic biology and other purposes. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 02/2015; 290(14). DOI:10.1074/jbc.M115.638148 · 4.57 Impact Factor
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    ABSTRACT: Over the past 30 years, multidrug resistant enterococci have emerged as leading causes of hospital acquired infection. With the acquisition of resistance to vancomycin in the mid 1980’s many of these infections became extremely difficult to treat. To address this problem, researchers with an interest in various aspects of enterococcal biology have come together to consolidate much of what is known about this genus into a single text. Enterococci: From commensals to leading causes of drug resistant infection provides state-of-the-art summaries of what is known about 1) Their origins, distribution in nature and gut colonization, 2) Infection – history, incidence, and pathology, 3) Enterococci as indicators of contamination and in public policy, 4) Infection treatment and antibiotic resistance, 5) Pathogenesis and models of enterococcal infection, 6) Comparative enterococcal genomics, 7) Nature, maintenance and transmission of extrachromosomal elements, 8) Enterococcal phage and genome defense, 9) Transcriptional and post transcriptional control of enterococcal gene regulation, 10) Enterococcal cell wall components and structures, 11) Enterococcal biofilm structure and role in colonization and disease, 12) Metabolic and physiologic traits that contribute to the special biology of enterococci, and 13) Enterococcal bacteriocins and other bacterial factors that contribute to niche control.
    Second edited by In: Gilmore MS, Clewell DB, Ike Y, Shankar N., 02/2014; Enterococci: From Commensals to Leading Causes of Drug Resistant Infection [Internet].
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    Finbarr Hayes · Barbara Kędzierska ·
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    ABSTRACT: Genes for toxin-antitoxin (TA) complexes are widely disseminated in bacteria, including in pathogenic and antibiotic resistant species. The toxins are liberated from association with the cognate antitoxins by certain physiological triggers to impair vital cellular functions. TAs also are implicated in antibiotic persistence, biofilm formation, and bacteriophage resistance. Among the ever increasing number of TA modules that have been identified, the most numerous are complexes in which both toxin and antitoxin are proteins. Transcriptional autoregulation of the operons encoding these complexes is key to ensuring balanced TA production and to prevent inadvertent toxin release. Control typically is exerted by binding of the antitoxin to regulatory sequences upstream of the operons. The toxin protein commonly works as a transcriptional corepressor that remodels and stabilizes the antitoxin. However, there are notable exceptions to this paradigm. Moreover, it is becoming clear that TA complexes often form one strand in an interconnected web of stress responses suggesting that their transcriptional regulation may prove to be more intricate than currently understood. Furthermore, interference with TA gene transcriptional autoregulation holds considerable promise as a novel antibacterial strategy: artificial release of the toxin factor using designer drugs is a potential approach to induce bacterial suicide from within.
    Toxins 01/2014; 6(1):337-58. DOI:10.3390/toxins6010337 · 2.94 Impact Factor
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    ABSTRACT: Extrachromosomal elements are ubiquitous in the prokaryotic world and play important roles in the adaptation and survival of cell populations, especially in changing environments. Plasmids are readily found in enterococci, and it is not unusual for clinical and commensal strains (e.g. Enterococcus faecalis and Enterococcus faecium) to harbor a number of such elements. Indeed, plasmid-free isolates are only infrequently identified. Enterococcal plasmids commonly encode: i) resistance to one or more antibiotics; ii) elevated resistance to ultraviolet light; iii) virulence factors, such as cytolysin and aggregation substance; and iv) bacteriocins. In addition, intercellular transmissibility is frequently a plasmid-determined trait. As in many bacterial species, plasmids generally range in size from 3–4 kb to well over 100 kb and may be present at relatively low copy number (1–2 copies) or up to 20 or more per cell. Table 1 presents a list of enterococcal plasmids recently compiled by one of the authors (Teresa M. Coque). Conjugation is a primary means for intercellular DNA mobility in enterococci—natural transformation has never been reported, and information is only beginning to be reported with regard to transduction involving a bacteriophage (see Enterococcal bacteriophages and genome defense). Some conjugative plasmids transfer efficiently from donor to recipient in broth, whereas others transfer well only on solid surfaces. In the case of E. faecalis, peptide sex pheromones secreted by recipient cells induce conjugation-related mating functions, determined by certain plasmids (e.g. pAD1, pCF10, and a host of others). Another group of plasmids, such as pMG1 and related elements identified mainly in E. faecium, are also able to transfer efficiently in broth, but do not appear to make use of sex pheromones. A group of plasmids exemplified by pAMβ1 do not transfer well in broth, but are able to move if the cells are on a solid surface. Nonconjugative plasmids are also commonly present in enterococci, and some are readily mobilized by conjugative elements in trans or move via co-integration in some cases. Representatives of some of the above-noted elements have been sequenced, and studies relating to their transfer mechanisms have been published. In addition, reports relating to replication and partitioning provide significant information on the ways in which certain transmissible elements are maintained in their host. Other types of transmissible elements common in enterococci are the so-called conjugative transposons, which are exemplified by the Tn916 family. Usually found integrated in the chromosome, their movement involves an excision event that results in a non-replicative circular intermediate that is able to transfer conjugatively, followed by insertion into the genome of a recipient cell. Originally identified in E. faecalis, these elements, which commonly encode antibiotic resistance traits, have a broad host range and are widespread among numerous bacterial genera. In a similar vein and as found to be the case for many species of bacteria in recent years, enterococci have been shown to carry a plethora of “genomic islands,” some of which are mobile and called “integrative conjugative elements” (ICEs). Some of these represent “pathogenicity islands” that confer significant virulence traits and even antibiotic resistance. Rapidly accumulating genomic sequencing data are facilitating identification of the enterococcal “mobilome,” which includes not only transmissible elements, but also insertion sequences, transposons, and integrons that move intracellularly. Studies based on functionality, including replication and maintenance, complement this rapidly expanding picture, and the significant extent to which enterococci have participated in horizontal transfer within the bacterial world is becoming readily apparent. Below we attempt to summarize recent developments in various aspects of mobile genetic elements (MGEs) in enterococci and try to provide a perspective that is relevant to bacterial-human interaction.
    Enterococci: From Commensals to Leading Causes of Drug Resistant Infection, Edited by Michael S Gilmore, Don B Clewell, Yasuyoshi Ike, Nathan Shankar, 01/2014; Massachusetts Eye and Ear Infirmary.
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    ABSTRACT: Toxin-antitoxin complexes are ubiquitous in bacteria. The specificity of interactions between toxins and antitoxins from homologous but non-interacting systems was investigated. Based on molecular modeling, selected amino acid residues were changed to assess which positions were crucial in specificity of toxin-antitoxin interaction in the related Axe-Txe and YefM-YoeB complexes. No cross-interactions between wild-type proteins was detected. However, a single amino acid substitution that converts a Txe-specific residue to a YoeB-specific residue reduced, but did not abolish, Txe interaction with the Axe antitoxin. Interestingly, this alteration (Txe-Asp83Tyr) promoted functional interactions between Txe and the YefM antitoxin. The interactions between Txe-Asp83Tyr and YefM were sufficiently strong to abolish Txe toxicity and to allow effective co-repression by YefM-Txe-Asp83Tyr of the promoter from which yefM-yoeB is expressed. We conclude that Asp83 in Txe is crucial for specificity of toxin-antitoxin interactions in the Axe-Txe complex, and that swapping this residue for the equivalent residue in YoeB relaxes the specificity of the toxin-antitoxin interaction. This article is protected by copyright. All rights reserved.
    FEBS Journal 09/2013; 280(22). DOI:10.1111/febs.12517 · 4.00 Impact Factor
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    ABSTRACT: Multidrug-resistant variants of human pathogens from the genus Enterococcus represent a significant health threat as leading agents of nosocomial infections. The easy acquisition of plasmid-borne genes is intimately involved in the spread of antibiotic resistance in enterococci. Toxin-antitoxin (TA) systems play a major role in both maintenance of mobile genetic elements that specify antibiotic resistance, and in bacterial persistence and virulence. Expression of toxin and antitoxin genes must be in balance as inappropriate levels of toxin can be dangerous to the host. The controlled production of toxin and antitoxin is usually achieved by transcriptional autoregulation of TA operons. One of the most prevalent TA modules in enterococcal species is axe-txe which is detected in a majority of clinical isolates. Here, we demonstrate that the axe-txe cassette presents a complex pattern of gene expression regulation. Axe-Txe cooperatively autorepress expression from a major promoter upstream of the cassette. However, an internal promoter that drives the production of a newly discovered transcript from within axe gene combined with a possible modulation in mRNA stability play important roles in the modulation of Axe:Txe ratio to ensure controlled release of the toxin.
    PLoS ONE 09/2013; 8(9):e73569. DOI:10.1371/journal.pone.0073569 · 3.23 Impact Factor
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    ABSTRACT: DNA segregation in bacteria is mediated most frequently by proteins of the ParA superfamily that transport DNA molecules attached via the segrosome nucleoprotein complex. Segregation is governed by a cycle of ATP-induced polymerization and subsequent depolymerization of the ParA factor. Here, we establish that hyperactive ATPase variants of the ParA homolog, ParF, display altered segrosome dynamics that block accurate DNA segregation. An arginine finger-like motif in the ParG centromere binding factor augments ParF ATPase activity, but is ineffective in stimulating nucleotide hydrolysis by the hyperactive proteins. Moreover, whereas polymerization of wild-type ParF is accelerated by ATP and inhibited by ADP, filamentation of the mutated proteins is blocked indiscriminately by nucleotides. The mutations affect a triplet of conserved residues that are situated neither in canonical nucleotide binding and hydrolysis motifs in the ParF tertiary structure nor at interfaces implicated in ParF polymerization. Instead the residues are involved in shaping the contours of the binding pocket so that nucleotide binding locks the mutant proteins into a configuration that is refractory to polymerization. Thus, the architecture of the pocket is crucial not only for optimal ATPase kinetics, but also plays a key role in the polymerization dynamics of ParA proteins that drive DNA segregation ubiquitously in procaryotes.
    Journal of Biological Chemistry 10/2012; 287(51). DOI:10.1074/jbc.M112.410324 · 4.57 Impact Factor
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    ABSTRACT: Segregation of the bacterial multidrug resistance plasmid TP228 requires the centromere-binding protein ParG, the parH centromere, and the Walker box ATPase ParF. The cycling of ParF between ADP- and ATP-bound states drives TP228 partition; ATP binding stimulates ParF polymerization, which is essential for segregation, whereas ADP binding antagonizes polymerization and inhibits DNA partition. The molecular mechanism involved in this adenine nucleotide switch is unclear. Moreover, it is unknown how any Walker box protein polymerizes in an ATP-dependent manner. Here, we describe multiple ParF structures in ADP- and phosphomethylphosphonic acid adenylate ester (AMPPCP)-bound states. ParF-ADP is monomeric but dimerizes when complexed with AMPPCP. Strikingly, in ParF-AMPPCP structures, the dimers interact to create dimer-of-dimer "units" that generate a specific linear filament. Mutation of interface residues prevents both polymerization and DNA segregation in vivo. Thus, these data provide insight into a unique mechanism by which a Walker box protein forms polymers that involves the generation of ATP-induced dimer-of-dimer building blocks.
    Journal of Biological Chemistry 06/2012; 287(31):26146-54. DOI:10.1074/jbc.M112.373696 · 4.57 Impact Factor
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    Massimiliano Zampini · Finbarr Hayes ·
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    ABSTRACT: Artificial transcription factors (ATFs) are potent synthetic biology tools for modulating endogenous gene expression and precision genome editing. The ribbon-helix-helix (RHH) superfamily of transcription factors are widespread in bacteria and archaea. The principal DNA binding determinant in this family comprises a two-stranded antiparallel β-sheet (ribbons) in which a pair of eight-residue motifs insert into the major groove. Here, we demonstrate that ribbons of divergent RHH proteins are compact and portable elements that can be grafted into a common α-helical scaffold producing active ATFs. Hybrid proteins cooperatively recognize DNA sites possessing core tetramer boxes whose functional spacing is dictated by interactions between the α-helical backbones. These interactions also promote combinatorial binding of chimeras with different transplanted ribbons, but identical backbones, to synthetic sites bearing cognate boxes for each protein either in vitro or in vivo. The composite assembly of interacting hybrid proteins offers potential advantages associated with combinatorial approaches to DNA recognition compared with ATFs that involve binding of a single protein. Moreover, the new class of RHH ATFs may be utilized to re-engineer transcriptional circuits, or may be enhanced with affinity tags, fluorescent moieties or other elements for targeted genome marking and manipulation in bacteria and archaea.
    Nucleic Acids Research 04/2012; 40(14):6673-82. DOI:10.1093/nar/gks314 · 9.11 Impact Factor
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    Finbarr Hayes · Laurence Van Melderen ·
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    ABSTRACT: Genes for toxin-antitoxin (TA) complexes are widespread in prokaryote genomes, and species frequently possess tens of plasmid and chromosomal TA loci. The complexes are categorized into three types based on genetic organization and mode of action. The toxins universally are proteins directed against specific intracellular targets, whereas the antitoxins are either proteins or small RNAs that neutralize the toxin or inhibit toxin synthesis. Within the three types of complex, there has been extensive evolutionary shuffling of toxin and antitoxin genes leading to considerable diversity in TA combinations. The intracellular targets of the protein toxins similarly are varied. Numerous toxins, many of which are sequence-specific endoribonucleases, dampen protein synthesis levels in response to a range of stress and nutritional stimuli. Key resources are conserved as a result ensuring the survival of individual cells and therefore the bacterial population. The toxin effects generally are transient and reversible permitting a set of dynamic, tunable responses that reflect environmental conditions. Moreover, by harboring multiple toxins that intercede in protein synthesis in response to different physiological cues, bacteria potentially sense an assortment of metabolic perturbations that are channeled through different TA complexes. Other toxins interfere with the action of topoisomersases, cell wall assembly, or cytoskeletal structures. TAs also play important roles in bacterial persistence, biofilm formation and multidrug tolerance, and have considerable potential both as new components of the genetic toolbox and as targets for novel antibacterial drugs.
    Critical Reviews in Biochemistry and Molecular Biology 08/2011; 46(5):386-408. DOI:10.3109/10409238.2011.600437 · 7.71 Impact Factor
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    ABSTRACT: Extracellular death factor is a quorum-sensing pentapeptide that relays cell density information to an intracellular toxin-antitoxin complex. In this issue of Molecular Cell, Belitsky et al. (2011) demonstrate that the peptide competes with the antitoxin for toxin binding and directly activates the latter's endoribonuclease activity.
    Molecular cell 03/2011; 41(6):617-8. DOI:10.1016/j.molcel.2011.02.032 · 14.02 Impact Factor
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    ABSTRACT: The segrosome of multiresistance plasmid TP228 comprises ParF, which is a member of the ParA ATPase superfamily, and the ParG ribbon-helix-helix factor that assemble jointly on the parH centromere. Here we demonstrate that the distinctive parH site (∼100-bp) consists of an array of degenerate tetramer boxes interspersed by AT-rich spacers. Although numerous consecutive AT-steps are suggestive of inherent curvature, parH lacks an intrinsic bend. Sequential deletion of parH tetramers progressively reduced centromere function. Nevertheless, the variant subsites could be rearranged in different geometries that accommodated centromere activity effectively revealing that the site is highly elastic in vivo. ParG cooperatively coated parH: proper centromere binding necessitated the protein's N-terminal flexible tails which modulate the centromere binding affinity of ParG. Interaction of the ParG ribbon-helix-helix domain with major groove bases in the tetramer boxes likely provides direct readout of the centromere. In contrast, the AT-rich spacers may be implicated in indirect readout that mediates cooperativity between ParG dimers assembled on adjacent boxes. ParF alone does not bind parH but instead loads into the segrosome interactively with ParG, thereby subtly altering centromere conformation. Assembly of ParF into the complex requires the N-terminal flexible tails in ParG that are contacted by ParF.
    Nucleic Acids Research 03/2011; 39(12):5082-97. DOI:10.1093/nar/gkr115 · 9.11 Impact Factor
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    ABSTRACT: RA3 is a low-copy-number, broad-host-range (BHR) conjugative plasmid of the IncU incompatibility group isolated originally from Aeromonas spp. A 4.9-kb fragment of RA3 is sufficient to stabilize an otherwise unstable replicon in Escherichia coli. This fragment specifies the korA-incC-korB-orf11 operon coding for an active partition system related to the central control operon of IncP-1 plasmids and found also in BHR environmental plasmids recently classified as the PromA group. All four genes in the cassette are necessary for segregation. IncC and KorB of RA3 belong to the ParA and ParB families of partitioning proteins, respectively. In contrast with IncP-1 plasmids, neither KorB nor IncC are involved in transcriptional autoregulation. Instead, KorA exerts transcriptional control of the operon by binding to a palindromic sequence that overlaps the putative −35 promoter motif of the cassette. The Orf11 protein is not required for regulation, but its absence decreases the stabilization potential of the segregation module. A region discontiguous from the cassette harbors a set of unrelated repeat motifs distributed over ∼300 bp. Dissection of this region identified the centromere sequence that is vital for partitioning. The ∼300-bp fragment also encompasses the origin of conjugative transfer, oriT, and the promoter that drives transcription of the conjugative transfer operon. A similar set of cis-acting motifs are evident in the PromA group of environmental plasmids, highlighting a common evolutionary origin of segregation and conjugative transfer modules in these plasmids and members of the IncU group.
    Applied and Environmental Microbiology 02/2011; 77(7):2414-27. DOI:10.1128/AEM.02338-10 · 3.67 Impact Factor
  • Finbarr Hayes · Daniela Barillà ·
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    ABSTRACT: Plasmids are extrachromosomal elements that are widely distributed in eubacteria, as well as in archaea and lower eukaryotes. Plasmids confer additional genetic plasticity on species that harbour them, but also are of major clinical significance because antibiotic resistance, virulence, and other disease-associated genes often reside on these highly mobile elements. Moreover, plasmids are malleable and informative models to improve understanding of bacterial genome segregation: the molecular mechanisms of bacterial DNA segregation are best described for low copy number plasmids. The segrosome is the nucleoprotein complex that drives accurate plasmid partitioning. The complex typically includes: (i) a centromere analogue on which segrosome assembly occurs; (ii) one of a diverse array of site-specific DNA binding factors that recognizes its cognate centromere and with which it forms a nucleoprotein structure of specific architecture; and (iii) an ATP binding protein, either actin-like or, more commonly, a Walker-type ATPase of the ParA superfamily that is unique to prokaryotes and which assembles into the mature segrosome. ATP-mediated polymerization of actin-like segregation proteins into a bipolar spindle elicits bidirectional filament growth, propelling attached plasmids in opposing directions prior to cytokinesis. Plasmid-encoded ParA proteins also polymerize in response to ATP binding, although the molecular mechanisms that underpin this behaviour and how this polymerization mediates intracellular plasmid trafficking remain to be fully elucidated. Recent insightful biochemical, structural and cell biological analyses of segrosome assembly and action continue to unravel fundamental aspects of plasmid segregation. KeywordsPlasmid-segregation-partition-ParA/actin
    12/2009: pages 49-70;
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    ABSTRACT: The segrosome is the nucleoprotein complex that mediates accurate plasmid segregation. In addition to its multifunctional role in segrosome assembly, the ParG protein of multiresistance plasmid TP228 is a transcriptional repressor of the parFG partition genes. ParG is a homodimeric DNA binding protein, with C-terminal regions that interlock into a ribbon-helix-helix fold. Antiparallel beta-strands in this fold are presumed to insert into the O(F) operator major groove to exert transcriptional control as established for other ribbon-helix-helix factors. The O(F) locus comprises eight degenerate tetramer boxes arranged in a combination of direct and inverted orientation. Each tetramer motif likely recruits one ParG dimer, implying that the fully bound operator is cooperatively coated by up to eight dimers. O(F) was subdivided experimentally into four overlapping 20-bp sites (A to D), each of which comprises two tetramer boxes separated by AT-rich spacers. Extensive interaction studies demonstrated that sites A to D individually are bound with different affinities by ParG (C > A approximately B > D). Moreover, comprehensive scanning mutagenesis revealed the contribution of each position in the site core and flanking sequences to ParG binding. Natural variations in the tetramer box motifs and in the interbox spacers, as well as in flanking sequences, each influence ParG binding. The O(F) operator apparently has evolved with sites that bind ParG dissimilarly to produce a nucleoprotein complex fine-tuned for optimal interaction with the transcription machinery. The association of other ribbon-helix-helix proteins with complex recognition sites similarly may be modulated by natural sequence variations between subsites.
    Journal of bacteriology 04/2009; 191(12):3832-41. DOI:10.1128/JB.01630-08 · 2.81 Impact Factor
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    Simon E S Bailey · Finbarr Hayes ·
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    ABSTRACT: YefM-YoeB is among the most prevalent and well-characterized toxin-antitoxin complexes. YoeB toxin is an endoribonuclease whose activity is inhibited by YefM antitoxin. The regions 5′ of yefM-yoeB in diverse bacteria possess conserved sequence motifs that mediate transcriptional autorepression. The yefM-yoeB operator site arrangement is exemplified in Escherichia coli: a pair of palindromes with core hexamer motifs and a center-to-center distance of 12 bp overlap the yefM-yoeB promoter. YefM is an autorepressor that initially recognizes a long palindrome containing the core hexamer, followed by binding to a short repeat. YoeB corepressor greatly enhances the YefM-operator interaction. Scanning mutagenesis demonstrated that the short repeat is crucial for correct interaction of YefM-YoeB with the operator site in vivo and in vitro. Moreover, altering the relative positions of the two palindromes on the DNA helix abrogated YefM-YoeB cooperative interactions with the repeats: complex binding to the long repeat was maintained but was perturbed to the short repeat. Although YefM lacks a canonical DNA binding motif, dual conserved arginine residues embedded in a basic patch of the protein are crucial for operator recognition. Deciphering the molecular basis of toxin-antitoxin transcriptional control will provide key insights into toxin-antitoxin activation and function.
    Journal of bacteriology 12/2008; 191(3):762-72. DOI:10.1128/JB.01331-08 · 2.81 Impact Factor
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    ABSTRACT: IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha- and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta- and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids.
    Applied and Environmental Microbiology 08/2008; 74(13):4119-32. DOI:10.1128/AEM.00229-08 · 3.67 Impact Factor
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    ABSTRACT: Multidrug-resistant variants of the opportunistic human pathogen Enterococcus have recently emerged as leading agents of nosocomial infection. The acquisition of plasmid-borne resistance genes is a driving force in antibiotic-resistance evolution in enterococci. The segregation locus of a high-level gentamicin-resistance plasmid, pGENT, in Enterococcus faecium was identified and dissected. This locus includes overlapping genes encoding PrgP, a member of the ParA superfamily of segregation proteins, and PrgO, a site-specific DNA binding homodimer that recognizes the cenE centromere upstream of prgPO. The centromere has a distinctive organization comprising three subsites, CESII separates CESI and CESIII, each of which harbors seven TATA boxes spaced by half-helical turns. PrgO independently binds both CESI and CESIII, but with different affinities. The topography of the complex was probed by atomic force microscopy, revealing discrete PrgO foci positioned asymmetrically at the CESI and CESIII subsites. Bending analysis demonstrated that cenE is intrinsically curved. The organization of the cenE site and of certain other plasmid centromeres mirrors that of yeast centromeres, which may reflect a common architectural requirement during assembly of the mitotic apparatus in yeast and bacteria. Moreover, segregation modules homologous to that of pGENT are widely disseminated on vancomycin and other resistance plasmids in enterococci. An improved understanding of segrosome assembly may highlight new interventions geared toward combating antibiotic resistance in these insidious pathogens.
    Proceedings of the National Academy of Sciences 03/2008; 105(6):2151-6. DOI:10.1073/pnas.0704681105 · 9.67 Impact Factor
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    ABSTRACT: The segrosome is the nucleoprotein complex that mediates accurate segregation of bacterial plasmids. The segrosome of plasmid TP228 comprises ParF and ParG proteins that assemble on the parH centromere. ParF, which exemplifies one clade of the ubiquitous ParA superfamily of segregation proteins, polymerizes extensively in response to ATP binding. Polymerization is modulated by the ParG centromere binding factor (CBF). The segrosomes of plasmids pTAR, pVT745 and pB171 include ParA homologues of the ParF subgroup, as well as diverse homodimeric CBFs with no primary sequence similarity to ParG, or each other. Centromere binding by these analogues is largely specific. Here, we establish that the ParF homologues of pTAR and pB171 filament modestly with ATP, and that nucleotide hydrolysis is not required for this polymerization, which is more prodigious when the cognate CBF is also present. By contrast, the ParF homologue of plasmid pVT745 did not respond appreciably to ATP alone, but polymerized extensively in the presence of both its cognate CBF and ATP. The co-factors also stimulated nucleotide-independent polymerization of cognate ParF proteins. Moreover, apart from the CBF of pTAR, the disparate ParG analogues promoted polymerization of non-cognate ParF proteins suggesting that filamentation of the ParF proteins is enhanced by a common mechanism. Like ParG, the co-factors may be modular, possessing a centromere-specific interaction domain linked to a flexible region containing determinants that promiscuously stimulate ParF polymerization. The CBFs appear to function as bacterial analogues of formins, microtubule-associated proteins or related ancillary factors that regulate eucaryotic cytoskeletal dynamics.
    Journal of Molecular Biology 12/2007; 374(1):1-8. DOI:10.1016/j.jmb.2007.09.025 · 4.33 Impact Factor

Publication Stats

2k Citations
330.13 Total Impact Points


  • 2001-2014
    • The University of Manchester
      • • Manchester Interdisciplinary Biocentre (MIB)
      • • Faculty of Life Sciences
      Manchester, England, United Kingdom
  • 2011
    • Universität Kassel
      • Department of Genetics
      Cassel, Hesse, Germany
  • 2009
    • The University of York
      • Department of Biology
      York, England, United Kingdom
  • 1997-1999
    • University of Oxford
      • Department of Biochemistry
      Oxford, England, United Kingdom
  • 1994
    • NCI-Frederick
      Фредерик, Maryland, United States
  • 1993
    • Leidos Biomedical Research
      Maryland, United States
  • 1990-1993
    • University College Cork
      • Department of Microbiology
      Corcaigh, Munster, Ireland