Structural Biology of Bacterial Multidrug Resistance Gene Regulators

Multidrug resistance (mdr)1 can be defined broadly as the ability of a cell to survive ordinarily lethal doses of more than one drug. Clearly, such resistance is a critical problem in the treatment of fungal and bacterial infections and cancer. Four general, but nonexclusive, mechanisms give rise to multidrug resistance: 1) detoxification by enzymatic modification or cleavage of drug; 2) genetic alteration of the intra- or extracellular targets; 3) decreased permeability of the cell membrane; and 4) active drug extrusion by multidrug transporters. Paramount to our understanding of mdr is the issue of recognition of structurally dissimilar substrates and how drug binding effects function. In bacteria many multidrug transporters are regulated directly (locally) by transcription factors, which also bind the substrates of these transporters, i.e. the drug can act as a transcriptional coactivator or inducer. Multiple mdr transporter genes are also regulated globally by activators such as MarA that do not necessarily bind drugs (1). The regulators are of keen interest because they are more amenable to structural studies than the membrane-bound transporters and thus offer a greater chance to obtain high resolution views of multidrug binding. Moreover, the local gene regulators are equally interesting as their DNA complexes directly reveal the mechanism of mdrtransporter gene regulation. This minireview will summarize the structures of known bacterial mdr regulators. Because our focus is more structural the reader is referred to one of several recent reviews that discuss the more biological aspects of global and local mdr regulation (2-5)

Crystal Structure of MtaN, a Global Multidrug Transporter Gene Activator

MtaN (Multidrug Transporter Activation, N terminus) is a constitutive, transcriptionally active 109-residue truncation mutant, which contains only the N-terminal DNA-binding and dimerization domains of MerR family member Mta. The 2.75 Å resolution crystal structure of apo-MtaN reveals a winged helix-turn-helix protein with a protruding 8-turn helix (α5) that is involved in dimerization by the formation of an antiparallel coiled-coil. The hydrophobic core and helices α1 through α4 are structurally homologous to MerR family member BmrR bound to DNA, whereas one wing (Wing 1) is shifted. Differences between the orientation of α5 with respect to the core and the revolution of the antiparallel coiled-coil lead to significantly altered conformations of MtaN and BmrR dimers. These shifts result in a conformation of MtaN that appears to be incompatible with the transcription activation mechanism of BmrR and suggest that additional DNA-induced structural changes are necessary

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

Is tagging with visual implant elastomer a reliable technique for marking earthworms?

Visual implant elastomer (VIE) has recently been employed to investigate different aspects of earthworm ecology. However, a number of fundamental questions relating to the detection and positioning of the tag, its persistence and potential effects on earthworms remain unknown. Seven earthworm species belonging to three ecological groupings, with different pigmentation and burrowing behaviour, were tagged using different coloured VIE. External inspection after two days, one week and 1, 10 and 27 months were followed by preservation, dissection and internal inspection. Tags could be seen in living specimens to 27 months, and dissection revealed that in most cases they were lodged in the coelomic cavity, held in place by septa. However, over longer time periods (more than two years), the chlorogogenous tissue tended to bind to the tags and made external observation increasingly difficult. Migration of the VIE material towards the posterior of the earthworm and potential loss of the tag were only observed on rare occasions, and a recovery rate in excess of 98% was recorded. By introducing a reasonable amount of VIE into segments, just after the clitellum, this technique can become a valuable tool in earthworm ecology and life history studies, particularly in short-medium term laboratory and field experiments

The potential for linking cohort participants to official criminal records:a pilot study using the Avon Longitudinal Study of Parents and Children (ALSPAC)

Introduction: Linking longitudinal cohort resources with police-recorded records of criminal activity has the potential to inform public health style approaches to policing, and may reduce potential sources of bias from self-reported criminal data collected by cohort studies. A pilot linkage of police records to the Avon Longitudinal Study of Parents and Children (ALSPAC) allows us to consider the acceptability of this linkage, its utility as a data resource, differences in self-reported crime according to consent status for data linkage, and the appropriate governance mechanism to support such a linkage. Methods: We carried out a pilot study linking data from the ALSPAC birth cohort to Ministry of Justice (MoJ) records on criminal cautions and convictions. This pilot was conducted on a fully anonymous basis, meaning we cannot link the identified records to any participant or the wider information within the dataset. Using ALSPAC data, we used summary statistics to investigate differences in socio-economic background and self-reported criminal activity by consent status for crime linkage. We used MoJ records to identify the geographic and temporal concentration of criminality in the ALSPAC cohort. Results: We found that the linkage appears acceptable to participants (4% of the sample opted out), levels of criminal caution and conviction are high enough to support research, and that the majority of crimes occurred in Avon & Somerset (the policing area local to ALSPAC). Those who did not respond to consent requests had higher levels of self-reported criminal behaviour compared to participants who provided explicit consent. Conclusions: These findings suggest that data linkage in ALSPAC provides opportunities to study criminal behaviour and that linked individual-level records could provide robust research in the area. Our findings also suggest the potential for bias when only including participants who have explicitly consented to data linkage, highlighting the limitations of opt-in consent strategies

Promoter Recognition by a Complex of Spx and the C-Terminal Domain of the RNA Polymerase α Subunit

Spx, an ArsC (arsenate reductase) family member, is a global transcriptional regulator of the microbial stress response and is highly conserved amongst Gram-positive bacteria. Bacillus subtilis Spx protein exerts positive and negative control of transcription through its interaction with the C-terminal domain of the RNA polymerase (RNAP) alpha subunit (alphaCTD). Spx activates trxA (thioredoxin) and trxB (thioredoxin reductase) in response to thiol stress, and bears an N-terminal C10XXC13 redox disulfide center that is oxidized in active Spx.The structure of mutant Spx(C10S) showed a change in the conformation of helix alpha4. Amino acid substitutions R60E and K62E within and adjacent to helix alpha4 conferred defects in Spx-activated transcription but not Spx-dependent repression. Electrophoretic mobility-shift assays showed alphaCTD interaction with trxB promoter DNA, but addition of Spx generated a supershifted complex that was disrupted in the presence of reductant (DTT). Interaction of alphaCTD/Spx complex with promoter DNA required the cis-acting elements -45AGCA-42 and -34AGCG-31 of the trxB promoter. The Spx(G52R) mutant, defective in alphaCTD binding, did not interact with the alphaCTD-trxB complex. Spx(R60E) not only failed to complex with alphaCTD-trxB, but also disrupted alphaCTD-trxB DNA interaction.The results show that Spx and alphaCTD form a complex that recognizes the promoter DNA of an Spx-controlled gene. A conformational change during oxidation of Spx to the disulfide form likely alters the structure of Spx alpha helix alpha4, which contains residues that function in transcriptional activation and alphaCTD/Spx-promoter interaction. The results suggest that one of these residues, R60 of the alpha4 region of oxidized Spx, functions in alphaCTD/Spx-promoter contact but not in alphaCTD interaction

Glypican-1, phosphacan/receptor protein-tyrosine phosphatase-ζ/β and its ligand, tenascin-C, are expressed by neural stem cells and neural cells derived from embryonic stem cells

The heparan sulfate proteoglycan glypican-1, the chondroitin sulfate proteoglycan phosphacan/RPTP (receptor protein-tyrosine phosphatase)-ζ/β and the extracellular matrix protein tenascin-C were all found to be expressed by neural stem cells and by neural cells derived from them. Expression of proteoglycans and tenascin-C increased after retinoic acid induction of SSEA1-positive ES (embryonic stem) cells to nestin-positive neural stem cells, and after neural differentiation, proteoglycans and tenascin-C are expressed by both neurons and astrocytes, where they surround cell bodies and processes and in certain cases show distinctive expression patterns. With the exception of tenascin-C (whose expression may decrease somewhat), expression levels do not change noticeably during the following 2 weeks in culture. The significant expression, by neural stem cells and neurons and astrocytes derived from them, of two major heparan sulfate and chondroitin sulfate proteoglycans of nervous tissue and of tenascin-C, a high-affinity ligand of phosphacan/RPTP-ζ/β, indicates that an understanding of their specific functional roles in stem cell neurobiology will be important for the therapeutic application of this new technology in facilitating nervous tissue repair and regeneration