Biomedical subjects

Ronald A Skurray

Publications and source records attributed to Ronald A Skurray.

12 recordsLinked to original sources

PubMed2006

Glycine-rich transmembrane helix 10 in the staphylococcal tetracycline transporter TetA(K) lines a solvent-accessible channel.

The staphylococcal TetA(K) tetracycline exporter is classified within the major facilitator superfamily of transport proteins and contains 14 alpha-helical transmembrane segments (TMS). Using cysteine-scanning mutagenesis, 27 amino acid residues across and flanking putative TMS 10 of the TetA(K) transporter were individually replaced with cysteine. The level of solvent accessibility to each of the targeted amino acid positions was determined as a measure of fluorescein maleimide reactivity and demonstrated that TMS 10 of TetA(K) has a cytoplasmic boundary at G313 and is likely to extend from at least V298 on the periplasmic side. TMS 10 was found to be amphiphilic containing at least partially solvent accessible amino acid residues along the length of one helical face, suggesting that this helix may line a solvent-exposed channel. Functional analyses of these cysteine mutants demonstrated a significant role for a number of amino acid residues, including a predominance of glycine residues which were further analyzed by alanine substitution. These residues are postulated to allow interhelical interactions between TMS 10 and distal parts of TetA(K) that are likely to be required for the tetracycline transport mechanism in TetA(K) and may be a general feature required by bacterial tetracycline transporters for activity.

Antiporters↗

PubMed2006

Functional effects of intramembranous proline substitutions in the staphylococcal multidrug transporter QacA.

The QacA multidrug transporter is encoded on Staphylococcus aureus multidrug resistance plasmids and confers broad-range antimicrobial resistance to more than 30 monovalent and bivalent lipophilic, cationic compounds from at least 12 different chemical classes. QacA contains 10 proline residues predicted to be within transmembrane regions, several of which are conserved in related export proteins. Proline residues are classically known as helix-breakers and are highly represented within the transmembrane helices of membrane transport proteins, where they can mediate the formation of structures essential for protein stability and transport function. The importance of these 10 intramembranous proline residues for QacA-mediated transport function was determined by examining the functional effect of substituting these residues with glycine, alanine or serine. Several proline-substituted QacA mutants failed to confer high-level resistance to selected QacA substrates. However, no single proline mutation, including those at conserved positions, significantly disrupted QacA protein expression or QacA-mediated resistance to all representative substrates, suggesting that these residues are not essential for the formation of structures requisite to the QacA substrate transport mechanism.

Amino Acid Sequence↗

PubMed2006

Replication control of staphylococcal multiresistance plasmid pSK41: an antisense RNA mediates dual-level regulation of Rep expression.

Replication of staphylococcal multiresistance plasmid pSK41 is negatively regulated by the antisense transcript RNAI. pSK41 minireplicons bearing rnaI promoter (PrnaI) mutations exhibited dramatic increases in copy number, approximately 40-fold higher than the copy number for the wild-type replicon. The effects of RNAI mutations on expression of the replication initiator protein (Rep) were evaluated using transcriptional and translational fusions between the rep control region and the cat reporter gene. The results suggested that when PrnaI is disrupted, the amount of rep mRNA increases and it becomes derepressed for translation. These effects were reversed when RNAI was provided in trans, demonstrating that it is responsible for significant negative regulation at two levels, with the greatest repression exerted on rep translation initiation. Mutagenesis provided no evidence for RNAI-mediated transcriptional attenuation as a basis for the observed reduction in rep message associated with expression of RNAI. However, RNA secondary-structure predictions and supporting mutagenesis data suggest a novel mechanism for RNAI-mediated repression of rep translation initiation, where RNAI binding promotes a steric transition in the rep mRNA leader to an alternative thermodynamically stable stem-loop structure that sequesters the rep translation initiation region, thereby preventing translation.

Bacterial Proteins↗

PubMed2006

Microbial drug efflux proteins of the major facilitator superfamily.

Drug efflux proteins are widespread amongst microorganisms, including pathogens. They can contribute to both natural insensitivity to antibiotics and to emerging antibiotic resistance and so are potential targets for the development of new antibacterial drugs. The design of such drugs would be greatly facilitated by knowledge of the structures of these transport proteins, which are poorly understood, because of the difficulties of obtaining crystals of quality. We describe a structural genomics approach for the amplified expression, purification and characterisation of prokaryotic drug efflux proteins of the 'Major Facilitator Superfamily' (MFS) of transport proteins from Helicobacter pylori, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Bacillus subtilis, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides and Streptomyces coelicolor. The H. pylori putative drug resistance protein, HP1092, and the S. aureus QacA proteins are used as detailed examples. This strategy is an important step towards reproducible production of transport proteins for the screening of drug binding and for optimisation of crystallisation conditions to enable subsequent structure determination.

Amino Acid Sequence↗

PubMed2005

Role of transmembrane segment 10 in efflux mediated by the staphylococcal multidrug transport protein QacA.

The staphylococcal multidrug exporter QacA confers resistance to a wide range of structurally dissimilar monovalent and bivalent cationic antimicrobial compounds. To understand the functional importance of transmembrane segment 10, which is thought to be involved in substrate binding, cysteine-scanning mutagenesis was performed in which 35 amino acid residues in the putative transmembrane helix and its flanking regions were replaced in turn with cysteine. Solvent accessibility analysis of the introduced cysteine residues using fluorescein maleimide indicated that transmembrane segment 10 of QacA contains a 20-amino-acid hydrophobic core and may extend from Pro-309 to Ala-334. Phenotypic analysis and fluorimetric transport assays of these mutants showed that Gly-313 is important for the efflux of both monovalent and bivalent cationic substrates, whereas Asp-323 is only important for the efflux of bivalent substrates and probably forms part of the bivalent substrate-binding site(s) together with Met-319. Furthermore, the effects of N-ethyl-maleimide treatment on ethidium and 4',6-diamidino-2-phenylindole export mediated by the QacA mutants suggest that the face of transmembrane segment 10 that contains Asp-323 may also be close to the monovalent substrate-binding site(s), making this helix an integral component of the QacA multidrug-binding pocket.

Amino Acid Sequence↗

PubMed2004

Staphylococcus aureus multiresistance plasmid pSK41: analysis of the replication region, initiator protein binding and antisense RNA regulation.

The vast majority of large staphylococcal plasmids characterized to date appear to possess an evolutionarily common replication system, which has clearly had a major impact on the evolution of antimicrobial resistant staphylococci worldwide. Related systems have also been found in plasmids from other Gram-positive genera, including enterococci, streptococci and bacilli. The 46.4 kb plasmid pSK41 is the prototype of a family of conjugative staphylococcal multiresistance plasmids. The replication region of pSK41 encodes a protein product, Rep, which was shown to be essential for replication; mutations that truncated Rep could be complemented in trans. Rep was found to bind in vitro to four tandem repeat sequences located centrally within the rep coding region. An A + T-rich inverted repeat sequence upstream of rep was required for efficient replication, whereas no sequences downstream of rep were necessary. An antisense countertranscript, RNAI, encoded upstream of rep was identified and transcriptional start points for both RNAI and the rep-mRNA were defined.

Amino Acid Sequence↗

PubMed2003

Interactions of the QacR multidrug-binding protein with structurally diverse ligands: implications for the evolution of the binding pocket.

The QacR multidrug-binding repressor protein regulates the expression of the Staphylococcus aureus qacA gene, a multidrug resistance (MDR) locus that is prevalent in clinical isolates of this important human pathogen. In this paper we demonstrate that the range of structurally diverse compounds capable of inducing qacA transcription is significantly more varied than previously appreciated, particularly in relation to bivalent cations. For all of the newly identified inducing compounds, induction of qacAexpression was correlated with a matching ability to dissociate QacR from operator DNA. Development of a ligand-binding assay based on intrinsic tryptophan fluorescence permitted dissociation constants to be determined for the majority of known QacR ligands, with values ranging from 0.2 to 82 microM. High-affinity binding of a compound to QacR in vitro was not found to correlate very strongly with either its in vivo inducing abilities or its structure. The latter observation indicated that the QacR ligand-binding pocket appears to have evolved to accommodate a wide range of toxic hydrophobic cations, rather than a specific class of compound. Importantly, the antimicrobial ligands of QacR included several plant alkaloids that share structural similarities with synthetic MDR substrates. This is consistent with the suggestion that the qacA-qacR MDR locus was recently derived from genes that protect against natural antimicrobial compounds.

ATP-Binding Cassette Transporters↗

PubMed2003

Stable low-copy-number Staphylococcus aureus shuttle vectors.

A series of Staphylococcus aureus-Escherichia coli shuttle vectors were constructed which contained the replication and maintenance functions of the S. aureus theta-mode multiresistance plasmid pSK1. The utility of the newly constructed vectors was demonstrated by the successful cloning and expression of several genes that had previously proven difficult to express in S. aureus. Additional vectors which permit the generation of transcriptional and translational fusions to an S. aureus blaZ reporter gene were also produced and subsequently employed to determine the relative strengths in S. aureus of a number of promoters. By utilizing the theta-mode replication functions of pSK1, the shuttle vectors described largely avoided the segregational and structural stability problems frequently encountered with Gram-positive rolling-circle-based vectors. In addition, these plasmids represent vectors which are suitable for the analysis of genes in S. aureus at low copy number.

Bacterial Proteins↗

PubMed2003

A single gene on the staphylococcal multiresistance plasmid pSK1 encodes a novel partitioning system.

The orf245 gene is located immediately upstream of, and divergently transcribed from, the replication initiation gene, rep, of the Staphylococcus aureus multiresistance plasmid pSK1, and related genes have been found in association with a range of evolutionarily distinct replication genes on plasmids from various gram-positive genera. orf245 has been shown previously to extend the segregational stability of a pSK1 minireplicon. Here we describe an investigation into the basis of orf245-mediated stabilization. orf245 was not found to influence transcription of pSK1 rep, indicating that it is not directly involved in plasmid replication. This was confirmed by demonstrating that orf245 is able to enhance the segregational stability of heterologous theta- and rolling-circle-replicating replicons, suggesting that it encodes a plasmid maintenance function. Evidence inconsistent with postsegregational killing and multimer resolution mechanisms was obtained; however, the intergenic region upstream of orf245 was found to mediate orf245-dependent incompatibility, as would be expected if it encodes a cis-acting centromere-like site. Taken together, these findings implicate active partitioning as the probable basis of the activity of orf245, which is therefore redesignated par. Since it is unrelated to any gene known to play a role in plasmid segregation, it seems likely that pSK1 par potentially represents the prototype of a novel class of active partitioning systems that are distinguished by their capacity to enhance plasmid segregational stability via a single protein-encoding gene.

Amino Acid Sequence↗

PubMed2002

Structural basis for cooperative DNA binding by two dimers of the multidrug-binding protein QacR.

The Staphylococcus aureus multidrug-binding protein QacR represses transcription of the qacA multidrug transporter gene and is induced by multiple structurally dissimilar drugs. QacR is a member of the TetR/CamR family of transcriptional regulators, which share highly homologous N-terminal DNA-binding domains connected to seemingly non-homologous ligand-binding domains. Unlike other TetR members, which bind approximately 15 bp operators, QacR recognizes an unusually long 28 bp operator, IR1, which it appears to bind cooperatively. To elucidate the DNA-binding mechanism of QacR, we determined the 2.90 A resolution crystal structure of a QacR-IR1 complex. Strikingly, our data reveal that the DNA recognition mode of QacR is distinct from TetR and involves the binding of a pair of QacR dimers. In this unique binding mode, recognition at each IR1 half-site is mediated by a complement of DNA contacts made by two helix-turn-helix motifs. The inferred cooperativity does not arise from cross-dimer protein-protein contacts, but from the global undertwisting and major groove widening elicited by the binding of two QacR dimers.

Bacterial Proteins↗

PubMed2002

Analysis and characterization of the IncFV plasmid pED208 transfer region.

pED208 is a transfer-derepressed mutant of the IncFV plasmid, F(0)lac, which has an IS2 element inserted in its traY gene, resulting in constitutive overexpression of its transfer (tra) region. The pED208 transfer region, which encodes proteins responsible for pilus synthesis and conjugative plasmid transfer, was sequenced and found to be very similar to the F tra region in terms of its organization although most pED208 tra proteins share only about 45% amino acid identity. All the essential genes for F transfer had homologs within the pED208 transfer region with the exception of traQ, which encodes the chaperone for stable F-pilin expression. F(0)lac appears to have a fertility inhibition system different than the FinOP system of other F-like plasmids, and its transfer efficiency was increased in the presence of F or R100, suggesting that it could be mobilized by these plasmids. The F-like transfer systems specified by F, R100, and F(0)lac were highly specific for their cognate origins of transfer (oriT) as measured by their abilities to mobilize chimeric oriT-containing plasmids.

Amino Acid Sequence↗

PubMed2002

Regulation of bacterial drug export systems.

The active transport of toxic compounds by membrane-bound efflux proteins is becoming an increasingly frequent mechanism by which cells exhibit resistance to therapeutic drugs. This review examines the regulation of bacterial drug efflux systems, which occurs primarily at the level of transcription. Investigations into these regulatory networks have yielded a substantial volume of information that has either not been forthcoming from or complements that obtained by analysis of the transport proteins themselves. Several local regulatory proteins, including the activator BmrR from Bacillus subtilis and the repressors QacR from Staphylococcus aureus and TetR and EmrR from Escherichia coli, have been shown to mediate increases in the expression of drug efflux genes by directly sensing the presence of the toxic substrates exported by their cognate pump. This ability to bind transporter substrates has permitted detailed structural information to be gathered on protein-antimicrobial agent-ligand interactions. In addition, bacterial multidrug efflux determinants are frequently controlled at a global level and may belong to stress response regulons such as E. coli mar, expression of which is controlled by the MarA and MarR proteins. However, many regulatory systems are ill-adapted for detecting the presence of toxic pump substrates and instead are likely to respond to alternative signals related to unidentified physiological roles of the transporter. Hence, in a number of important pathogens, regulatory mutations that result in drug transporter overexpression and concomitant elevated antimicrobial resistance are often observed.

Bacterial Proteins↗