The staphylococcal QacR multidrug regulator binds a correctly spaced operator as a pair of dimers

Expression of the Staphylococcus aureus plasmid-encoded QacA multidrug transporter is regulated by the divergently encoded QacR repressor protein. To circumvent the formation of disulfide-bonded degradation products, site-directed mutagenesis to replace the two cysteine residues in wild-type QacR was undertaken. Analysis of a resultant cysteineless QacR derivative indicated that it retained full DNA-binding activities in vivo and in vitro and continued to be fully proficient for the mediation of induction of qacA expression in response to a range of structurally dissimilar multidrug transporter substrates. The cysteineless QacR protein was used in cross-linking and dynamic light-scattering experiments to show that its native form was a dimer, whereas gel filtration indicated that four QacR molecules bound per DNA operator site. The addition of inducing compounds led to the dissociation of the four operator-bound QacR molecules from the DNA as dimers. Binding of QacR dimers to DNA was found to be dependent on the correct spacing of the operator half-sites. A revised model proposed for the regulation of qacA expression by QacR features the unusual characteristic of one dimer of the regulatory protein binding to each operator half-site by a process that does not appear to require the prior self-assembly of QacR into tetramers

Physiology of Escherichia coli K-12 during conjugation / Ronald A. Skurray.

Reprints to two articles published by the author held in pocket in back of publicationx, 158, xxvi leaves : ill. ; 26 cm.Thesis (Ph.D.) -- University of Adelaide, Dept. of Microbiology, 197

Molecular genetics of bacterial plasmids and transposons : collected reprints 1973-1989

1 v.Title page, contents and abstract only. The complete thesis in print form is available from the University Library.Thesis (D.Sc.)--University of Adelaide, 199

Staphyloccal multidrug efflux protein QacA

The QacA multidrug exporter from Staphylococcus aureus mediates resistance to a wide array of monovalent or divalent cationic, lipophilic, antimicrobial compounds. QacA provides resistance to these various compounds via a proton motive forcedependent antiport mechanism that conforms to classical Michaelis-Menten kinetics. Fluorescent transport analyses have demonstrated that this QacA:substrate interaction occurs with high affinity and competition studies have shown that QacAmediated ethidium export is competitively inhibited by other monovalent cations, and non-competitively inhibited by divalent cations, suggesting that monovalent and divalent cations bind at distinct sites on the QacA protein. The closely related export protein QacB, mediates lower levels of resistance to divalent cations, and lacks a high affinity-binding site for divalent cations. The cell membrane has been identified as the origin of QacA-mediated efflux; substrates are bound and expelled from within this hydrophobic environment. Regulation of qacA expression is achieved via the trans-acting repressor protein, QacR. QacR belongs to the TetR family of transcriptional repressor proteins, which all possess a helix-turn-helix DNA-binding domain at their N-terminal ends, and have highly divergent C-termini postulated to be involved in the binding of inducing compounds. QacR specifically binds to an inverted repeat, IR1, which has been identified as the qacA operator region, and overlaps the identified promoter sequence for qacA. QacR, like the multidrug export protein whose expression it regulates, has been shown to interact directly with a number of structurally-dissimilar compounds

Identification of suitable internal controls to study expression of a Staphylococcus aureus multidrug resistance system by quantitative real-time PCR

Quantitative real-time PCR (qRT-PCR) has become a routine technique for gene expression analysis. Housekeeping genes are customarily used as endogenous references for the relative quantification of genes of interest. The aim of this study was to develop a quantitative real-time PCR assay to analyze gene expression in multidrug resistant Staphylococcus aureus in the presence of cationic lipophilic substrates of multidrug transport proteins. Eleven different housekeeping genes were analyzed for their expression stability in the presence of a range of concentrations of four structurally different antimicrobial compounds. This analysis demonstrated that the genes rho, pyk and proC were least affected by rhodamine 6G and crystal violet, whereas fabD, tpiA and gyrA or fabD, proC and pyk were stably expressed in cultures grown in the presence of ethidium or berberine, respectively. Subsequently, these housekeeping genes were used as internal controls to analyze expression of the multidrug transport protein QacA and its transcriptional regulator QacR in the presence of the aforementioned compounds. Expression of qacA was induced by all four compounds, whereas qacR expression was found to be unaffected, reduced or enhanced. This study demonstrates that staphylococcal gene expression, including housekeeping genes previously used to normalize qRT-PCR data, is affected by growth in the presence of different antimicrobial compounds. Thus, identification of suitable genes usable as a control set requires rigorous testing. Identification of a such a set enabled them to be utilized as internal standards for accurate quantification of transcripts of the qac multidrug resistance system from S. aureus grown under different inducing conditions. Moreover, the qRT-PCR assay presented in this study may also be applied to gene expression studies of other multidrug transporters from S. aureus

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

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

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Optimized production and analysis of the staphylococcal multidrug efflux protein QacA

The plasmid-encoded QacA multidrug transport protein confers high-level resistance to a range of commonly used antimicrobials and is carried by widespread clinical strains of the human pathogen Staphylococcus aureus making it a potential target for future drug therapies. In order to obtain a sufficient yield of QacA protein for structural and biophysical studies, an optimized strategy for QacA overexpression was developed. QacA expression, directed from several vector systems in Escherichia coli, was tested under various growth and induction conditions and a synthetic qacA gene, codon-optimized for expression in E. coli was developed. Despite the extreme hydrophobicity and potential toxicity of the QacA secondary transport protein, a strategy based on the pBAD expression system, yielding up to four milligrams of approximately 95% pure QacA protein per litre of liquid culture, was devised. Purified QacA protein was examined using circular dichroism spectroscopy and displayed a secondary structure akin to that predicted from in silico analyses. Additionally, detergent solubilized QacA protein was shown to bind its fluorescent substrate rhodamine 6G with micro-molar affinity using a fluorescence polarization-based binding assay, similar to other multidrug transport proteins. To check the applicability of the expression/purification system described for QacA to other staphylococcal secondary transporters, the gene encoding the TetA(K) tetracycline efflux protein, which was previously recalcitrant to overexpression, was incorporated into the pBAD-based system and shown to be readily produced at easily detectable levels. Therefore, this expression system could be of general use for the production of secondary transport proteins in E. coli.7 page(s