Staphylococcus aureusA bout one-third of all proteins exploit specific metal ions to assist in macromolecular folding and͞or function at the active site of metalloenzymes (1). All cells restrict the number of bioavailable metal atoms to avoid any excess that would otherwise compete with native metal ion sites that do not support biological activity (2). Essentially all cell types contain intracellular metal sensors that detect surplus metal ions and control the expression of genes encoding proteins that expel or sequester the extra ions (3). For some metals and some cell types, a complementary set of sensors detect deficiency and regulate genes encoding proteins that acquire more of the required ions (4, 5). It is currently poorly understood how such metal-sensing metalloregulators accurately discriminate between various metal ions.SmtB͞ArsR-family regulators are ubiquitous in bacterial genomes and bind to the operator͞promoter (O͞P) regions of gene(s) encoding proteins involved in metal export or sequestration, repressing transcription (for a review, see ref. 6). As the concentration of metal ion increases, the effector-binding sites of the regulators become occupied eliciting a conformational change that weakens the affinity for the O͞P region, allowing transcription to proceed. Members of the SmtB͞ArsR family include: As(III), Sb(III), Bi(III)-responsive ArsR (7), Zn(II)-responsive SmtB (8), Cd(II), Pb(II), Bi(III)-responsive CadC (9-11), Zn(II)-responsive ZiaR (12), Co(II), Zn(II)-responsive CzrA (13,14), and, most recently, Ni(II), Co(II)-responsive NmtR (15).Comparative structural and spectroscopic studies of six SmtB͞ ArsR family members reveal that individual members are characterized by one or both of two structurally distinct metal coordination sites (6, 11, 15-20). These two metal sites are designated ␣3N (or ␣3) and ␣5 (or ␣5C), named for the location of the metal-binding ligands within the known or predicted secondary structure of individual family members. The coordination environment and precise ligand set of the ␣3, ␣3N, and͞or ␣5, ␣5C sites in the different SmtB͞ArsR proteins differ and are presumed to contribute toward metal selectivity. A sequence comparison for proteins discussed herein is shown in Fig. 1 and highlights these sites.Here we report insights gained from the study of two additional family members, Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtR. CzrA and NmtR share 30% sequence identity and a high degree of similarity (60%) yet respond to distinct but partially overlapping metal profiles in vivo. S. aureus CzrA is a Co(II)͞Zn(II)-specific sensor that regulates the expression of the czr operon, which encodes a Co(II)͞ Zn(II)-facilitated pump, CzrB, that effluxes metal out of the cell (13, 14). Electromobility-shift assays and in vivo expression studies indicate that Zn(II) is the strongest inducer of CzrA regulation, with Co(II) also capable of regulation but only at higher concentrations than Zn(II). Other metals, including Ni(II), have little to no effect on derep...
The mechanism of hydrosilylation using the highly active precatalyst Karstedt's precatalyst (Pt x -(M vinyl M vinyl ) y , M vinyl M vinyl ) divinyltetramethyldisiloxane) was investigated using extended X-ray absorption fine structure (EXAFS), small-angle X-ray scattering (SAXS), and ultraviolet-visible (UV-vis) spectroscopy. Contrary to previous reports suggesting colloidal catalysts, we find that regardless of the stoichiometric ratio of hydrosilane to olefin, the catalyst is a monomeric platinum compound with silicon and carbon in the first coordination sphere. The platinum end product, however, is a function of the stoichiometry of the reactants. At excess olefin concentration, the platinum end product contains only platinum-carbon bonds, whereas at high hydrosilane concentration, the platinum end product is multinuclear and also contains platinum-silicon bonds. The two end products can interconvert by adding additional aliquots of the stoichiometrically deficient reagent. An explanation of the "oxygen" effect is also given. In the absence of oxygen, hydrosilylation of certain olefins does not occur. Oxygen serves to disrupt multinuclear platinum species that are formed when poorly stabilizing olefins are employed. Finally, we discuss the olefin isomerization reaction that may accompany hydrosilylation of terminal olefins. When the hydrosilylation reaction proceeds slowly due to poorly reactive olefins, the olefin isomerization products become significant. EXAFS analysis of solutions after olefin isomerization has occurred shows the presence of platinum-platinum bonded compounds.
In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated single-and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of this important hyperthermophile.It would be difficult to overestimate the contribution of genetic manipulation to the study of any biological system, and it is an essential tool for the metabolic engineering of biosynthetic and substrate utilization pathways. This is particularly true for the archaea since, in spite of their environmental and industrial importance, coupled with their unique molecular features, much remains to be learned about their biology (2). The marine hyperthermophilic anaerobe Pyrococcus furiosus is of special interest not only for its ability to grow optimally at 100°C and the implications of this trait for its biology but also for industrial applications of its enzymes, as well as its capacity to produce hydrogen efficiently (4, 13, 44). The ability to apply genetic analyses of P. furiosus to underpin existing biochemical and molecular studies will contribute greatly to the establishment of P. furiosus as a model organism, particularly for biological hydrogen production.The development of genetic systems in the archaea, in general, presents many unique challenges given the extreme growth requirements of many of these organisms. To date, genetic systems of various levels of sophistication have been developed for representatives of all major groups of archaea, including halophiles, methanogens, thermoacidophiles, and hyperthermophiles (2,6,30,40,43,46). A variety of transformation methods are being used, including electroporation, heat shock with or without CaCl 2 treatment, phage-mediated transduction, spheroplast transformation, liposomes, and, very recently, even conjugation with Escherichia coli (2, 12). Transformation via natural competence has been reported in three archaeal species, in comparison to over 60 bacterial species that are known to exhibit this trait (16,36). Two of them are the methanogens Methanococcus voltae PS (7, 27) and Methanobacterium thermoautotrophicum Marburg (47); however, transformation frequencies were low, and there have been no follow-up studies regarding natural competence. The other is the hyperthermophile Thermococcus kodakarensis, which has an optimal growth temperature of 85°C. Its natural competence has enabled the development of genetic tools fo...
All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027-0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_ 0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes.The Gram-positive opportunistic human pathogen Staphylococcus aureus is the causative agent of a wide range of hospital and community-acquired infections that are associated with significant morbidity (1). With the incidence of methicillinresistant strains increasing in previously low prevalence areas (2), new antibiotic therapies that target novel metabolic pathways are urgently needed. One approach is to target those processes that allow a pathogen to respond to environmental stresses that might change depending on the microenvironmental host niche in which the organism finds itself. Resistance to host-mediated copper killing of Escherichia coli (3), Salmonella enterica (4), and Mycobacterium tuberculosis (5, 6) and sulfur assimilation and cysteine biosynthesis in M. tuberculosis (7,8) are two such processes. S. aureus is particularly sensitive to rapid killing when exposed to copper or copper alloy surfaces, justifying this therapeutic direction (9, 10).M. tuberculosis CsoR 6 (copper-sensitive operon repressor) is a founding member of large family of regulators now known collectively to respond to Cu(I), Ni(II), and perhaps other stressors, the structural basis of which is not fully understood (11, 12). All CsoR family proteins lack a known canonical DNA binding domain and are projected to adopt the flat disc-shaped dimer of dimers homotetrameric structure characteristic of Cu(I)-sensing CsoRs, with individual dimers consisting of an antiparallel four-helix bundle flanked by a C-terminal ␣3 helix (13,14). Two cysteine residues on opposite subunits within a dimer make coordination bonds to the Cu(I) ion, with the third ...
Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.
The Synechococcus PCC7942 SmtB is a zinc-responsive transcriptional repressor and a member of the ArsR superfamily of prokaryotic metalloregulatory transcription factors. The mechanism of negative regulation by Zn(II) and other metals as well as the coordination chemistry (stoichiometry, affinity, and specificity) of SmtB is poorly understood. In contrast to previous results [Kar, S. R., Adams, A. C., Lebowitz, J., Taylor, K. B., and Hall, L. M. (1997) Biochemistry 36, 15343-15348], we find that fully reduced SmtB binds 1 mol equiv of Zn(II) with a very high affinity, K(Zn) in excess of 10(11) M(-1) (pH 7.4, 0.15 M KCl, 22 degrees C). Optical spectroscopic experiments reveal that SmtB binds 1 mol equiv of Co(II) in a tetrahedral or distorted tetrahedral environment with one or two cysteine thiolate ligands in the first coordination shell. Zn(II) and Co(II) EXAFS studies are consistent with the optical spectroscopic data, and further suggest the presence of a mixture of carboxylate and imidazole-containing ligands. K(Co) was determined to be 1.7 (+/-0.1) x 10(9) M(-1) in a chelator (EGTA) competition assay; 1 equiv of Zn(II) results in complete displacement of the bound Co(II). SmtB also binds 1 mol equiv of Ni(II), which, when formed at low Ni(II):SmtB molar ratios, adopts a non-native, six-coordinate complex characterized by at least two histidine and no thiolate ligands. The hierarchy of metal binding affinities is Zn(II) >> Co(II) >> Ni(II).
Interest in the molecular structure of amyloid fibrils originates both from their association with many devastating diseases and as systems for exploring the energetics of higher order protein folding and assembly. These fibril arrays are generally viewed as rich in β-sheets, of either parallel or antiparallel orientation. [1][2][3][4][5] However, the relative arrangement of the sheets within the fibril remains poorly constrained in the existing structure models, 4,6,7 as these sheet-to-sheet arrangements are mediated predominantly by side chain packing. We now extend the use of metal ions as probes of amyloid side chain packing in simple segments of the Aβ peptide of Alzheimer's disease. By restricting the possible metal binding sites, we show that Zn 2+ can specifically control the rate of self-assembly and dramatically regulate amyloid morphology via distinct coordination environments.The histidine dyad, His13 and His14, of Aβ is implicated in metal binding 8,9 and the metalmediated toxicity of Aβ. [10][11][12][13] In a parallel, in-register β-sheet arrangement with sheet Hbonds oriented along the fibril axis (Figure 1a.), 3,4 the side chains of the His13 and His14 are spaced 5 Å apart along each surface of the β-sheets (Figure 1b). If the sheets are arrayed parallel to one another, the His13 and His14 side chains from different sheets are proximal, providing potential sites for Zn 2+ chelation along the sheets (Figure 1b), between the sheets (Figure 1c), or both. 6 Aβ(13-21), HHQKLVFFA, includes both the core segment, Aβ(17-21), known to be crucial for fibril formation, 14-18 and the metal binding dyad. To isolate His13/14 as the sole binding elements, the K16A peptide HHQALVFFA-NH 2 , Aβ(13-21)K16A, was prepared. As shown in Figure 2a, Aβ(13-21)K16A develops β-sheet secondary structure within 49 h, showing an increased mean residue molar ellipticity (MRME) by circular dichroism (CD) at 197 and 212 nm. The development of β-sheet structure was further confirmed by FTIR, showing the appearance of the amide I absorbance at 1628 cm −1 ( Figure S1a). TEM further established that Aβ(13-21)K16A assembles into fibrils (Fig. S1b), and these mature fibrils bind Congo red with the typical UV/vis absorption shift from 500 to 540 nm ( Figure S1c Figure 3). When the ribbons from the 1:1 incubation were pelleted, washed, and analyzed, the Zn 2+ to peptide ratio was 0.6-0.8 across three independent measurements. 19Wider 100-150 nm ribbons with variable twists form with longer incubation times (panels 2a, b, Figure 3). Some of the ribbons appear to coil and fuse to form tubular structures 200-300 nm in diameter (panels 2c, d, Figure 3). Therefore, Zn 2+ reduces the nucleation time of self-assembly across the entire concentration range and transforms Aβ(13-21)K16A assembly into either fibrillar or ribbon/ tubular morphology. 17 Struck by the different morphologies accessible to Aβ(13-21)-K16A, we investigated the coordination environment of Zn 2+ in the different assemblies by X-ray absorption spectroscopy (XAS). The soluble ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.