The Role of ß-Glycosylated Wall Teichoic Acids in the Reduction of Vancomycin Susceptibility in Vancomycin-Intermediate Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen that causes a wide range of infections. Due to the rapid evolution of antibiotic resistance that leads to treatment failure, it is important to understand the underlying mechanisms. Here, the cell wall structures of several laboratory vancomycin-intermediate S. aureus (VISA) strains were analyzed. Among the VISA strains were S. aureus VC40, which accumulated 79 mutations, including most importantly 2 exchanges in the histidine-kinase VraS, and developed full resistance against vancomycin (MIC, 64 µg/ml); a revertant S. aureus VC40R, which has an additional mutation in vraR (MIC, 4 µg/ml); and S. aureus VraS(VC40), in which the 2 vraS mutations were reconstituted into a susceptible background (MIC, 4 µg/ml). A ultraperformance liquid chromatography (UPLC) analysis showed that S. aureus VC40 had a significantly decreased cross-linking of the peptidoglycan. Both S. aureus VC40 and S. aureus VraS(VC40) displayed reduced autolysis and an altered autolysin profile in a zymogram. Most striking was the significant increase in d-alanine and N-acetyl-d-glucosamine (GlcNAc) substitution of the wall teichoic acids (WTAs) in S. aureus VC40. Nuclear magnetic resonance (NMR) analysis revealed that this strain had mostly ß-glycosylated WTAs in contrast to the other strains, which showed only the α-glycosylation peak. Salt stress induced the incorporation of ß-GlcNAc anomers and drastically increased the vancomycin MIC for S. aureus VC40R. In addition, ß-glycosylated WTAs decreased the binding affinity of AtlA, the major autolysin of S. aureus, to the cell wall, compared with α-glycosylated WTAs. In conclusion, there is a novel connection between wall teichoic acids, autolysis, and vancomycin susceptibility in S. aureus. IMPORTANCE Infections with methicillin-resistant Staphylococcus aureus are commonly treated with vancomycin. This antibiotic inhibits cell wall biosynthesis by binding to the cell wall building block lipid II. We set out to characterize the mechanisms leading to decreased vancomycin susceptibility in a laboratory-generated strain, S. aureus VC40. This strain has an altered cell wall architecture with a thick cell wall with low cross-linking, which provides decoy binding sites for vancomycin. The low cross-linking, necessary for this resistance mechanism, decreases the stability of the cell wall against lytic enzymes, which separate the daughter cells. Protection against these enzymes is provided by another cell wall polymer, the teichoic acids, which contain an unusually high substitution with sugars in the ß-conformation. By experimentally increasing the proportion of ß-N-acetyl-d-glucosamine in a closely related isolate through the induction of salt stress, we could show that the ß-conformation of the sugars plays a vital role in the resistance of S. aureus VC40.

High-affinity binding and catalytic activity of His/Tyr-based sequences: Extending heme-regulatory motifs beyond CP.

BACKGROUND & MOTIVATION: Peptides and proteins can interact with heme through His, Tyr, or Cys in heme-regulatory motifs (HRMs). The Cys-Pro dipeptide is a well investigated HRM, but for His and Tyr such a distinct motif is currently unknown. In addition, many heme-peptide complexes, such as heme-amyloid ß, can display a peroxidase-like activity, albeit there is little understanding of how the local primary and secondary coordination environment influences catalytic activity. We thus systematically evaluated a series of His- and Tyr-based peptides to identify sequence features for high-affinity heme binding and their impact on the catalytic activity of heme. METHODS: We employed solid-phase peptide synthesis to produce 58 nonapeptides, which were investigated by UV/vis, resonance Raman, and 2D NMR spectroscopy. A chromogenic assay was used to determine the catalytic activity of the heme-peptide complexes. RESULTS: Heme-binding affinity and binding mode were found to be dependent on the coordinating amino acid and spacer length between multiple potential coordination sites in a motif. In particular, HXH and HXXXH motifs showed strong heme binding. Analysis of the peroxidase-like activity revealed that some of these peptides and also HXXXY motifs enhance the catalytic activity of heme significantly. CONCLUSIONS: We identify HXH, HXXXH, and HXXXY as potential new HRMs with functional properties. Several peptides displayed a strikingly high peroxidase-like activity. GENERAL SIGNIFICANCE: The identification of HRMs allows to discover yet unknown heme-regulated proteins, and consequently, enhances our current understanding of pathologies involving labile heme.

Styrene Polymerization under Ambient Conditions by using a Transient 1,3,2-Diazaphospholane-2-oxyl Complex.

A combined theoretical and experimental study on the formation and reactivity of a P-OTEMP (P-bound TEMPO (TEMPO=2,2,6,6-tetramethyl-piperidin-1-oxyl)) substituted 1,3,2-diazaphospholane W(CO)5 complex is presented, including DFT-based mechanistic details. The complex possesses a thermally labile O-N bond that cleaves homolytically yielding the transient 1,3,2-diazaphospholane-2-oxyl complex [(CO)5 W(R2 PO. )], which acts as a radical initiator for styrene polymerization under ambient conditions.

The 5'-AG5 CC-3' Fragment from the Human CPEB3 Ribozyme Forms an Ultrastable Parallel RNA G-Quadruplex.

An unusually thermostable G-quadruplex is formed by a sequence fragment of a naturally occurring ribozyme, the human CPEB3 ribozyme. Strong evidence is provided for the formation of a uniquely stable intermolecular G-quadruplex structure consisting of five tetrad layers, by using CD spectroscopy, UV melting curves, 2D NMR spectroscopy, and gel shift analysis. The cationic porphyrin TMPyP4 destabilizes the complex.

Synthesis and Rearrangement of P-Nitroxyl-Substituted P(III) and P(V) Phosphanes: A Combined Experimental and Theoretical Case Study.

Low-temperature generation of P-nitroxyl phosphane 2 (Ph2 POTEMP), which was obtained by the reaction of Ph2 PH (1) with two equivalents of TEMPO, is presented. Upon warming, phosphane 2 decomposed to give P-nitroxyl phosphane P-oxide 3 (Ph2 P(O)OTEMP) as one of the final products. This facile synthetic protocol also enabled access to P-sulfide and P-borane derivatives 7 and 13, respectively, by using Ph2 P(S)H (6) or Ph2 P(BH3 )H (11) and TEMPO. Phosphane sulfide 7 revealed a rearrangement to phosphane oxide 8 (Ph2 P(O)STEMP) in CDCl3 at ambient temperature, whereas in THF, thermal decomposition of sulfide 7 yielded salt 10 ([TEMP-H2 ][Ph2 P(S)O]). As well as EPR and detailed NMR kinetic studies, indepth theoretical studies provided an insight into the reaction pathways and spin-density distributions of the reactive intermediates.

Multiple conformational states of riboswitches fine-tune gene regulation.

Riboswitches are structured regions of mRNAs that modulate gene expression in response to specific binding of low molecular-weight ligands. They function by induced transitions between different functional conformations. The standard model assumed that the two functional states, the ligand-bound and ligand-free state, populated only two stable conformations. Recent discoveries of multiple conformations for the apo-state and holo-state of riboswitches challenge this model. Moreover, it becomes evident that detected conformational heterogeneity--mostly in the apo-state--provides sensitivity to multiple environmental inputs for riboswitch-based gene-regulation.

The importance of helix P1 stability for structural pre-organization and ligand binding affinity of the adenine riboswitch aptamer domain.

We report here an in-depth characterization of the aptamer domain of the transcriptional adenine-sensing riboswitch (pbuE) by NMR and fluorescence spectroscopy. By NMR studies, the structure of two aptamer sequences with different lengths of the helix P1, the central element involved in riboswitch conformational switching, was characterized. Hydrogen-bond interactions could be mapped at nucleotide resolution providing information about secondary and tertiary structure, structure homogeneity and dynamics. Our study reveals that the elongation of helix P1 has pronounced effects not only on the local but on the global structure of the apo aptamer domain. The structural differences induced by stabilizing helix P1 were found to be linked to changes of the ligand binding affinity as revealed from analysis of kinetic and thermodynamic data obtained from stopped-flow fluorescence studies. The results provide new insight into the sequence-dependent fine tuning of the structure and function of purine-sensing riboswitches.

Three-state mechanism couples ligand and temperature sensing in riboswitches.

Riboswitches are cis-acting gene-regulatory RNA elements that can function at the level of transcription, translation and RNA cleavage. The commonly accepted molecular mechanism for riboswitch function proposes a ligand-dependent conformational switch between two mutually exclusive states. According to this mechanism, ligand binding to an aptamer domain induces an allosteric conformational switch of an expression platform, leading to activation or repression of ligand-related gene expression. However, many riboswitch properties cannot be explained by a pure two-state mechanism. Here we show that the regulation mechanism of the adenine-sensing riboswitch, encoded by the add gene on chromosome II of the human Gram-negative pathogenic bacterium Vibrio vulnificus, is notably different from a two-state switch mechanism in that it involves three distinct stable conformations. We characterized the temperature and Mg(2+) dependence of the population ratios of the three conformations and the kinetics of their interconversion at nucleotide resolution. The observed temperature dependence of a pre-equilibrium involving two structurally distinct ligand-free conformations of the add riboswitch conferred efficient regulation over a physiologically relevant temperature range. Such robust switching is a key requirement for gene regulation in bacteria that have to adapt to environments with varying temperatures. The translational adenine-sensing riboswitch represents the first example, to our knowledge, of a temperature-compensated regulatory RNA element.

Quantitative 2D and 3D Gamma-HCP experiments for the determination of the angles alpha and zeta in the phosphodiester backbone of oligonucleotides.

The quantitative Gamma-(HCP) experiment, a novel heteronuclear NMR pulse sequence for the determination of the RNA backbone angles alpha(O3'(i-1)-P(i)-O5'(i)-C5'(i)) and zeta(C3'(i)-O3'(i)-P(i+1)-O5'(i+1)) in (13)C-labeled RNA, is introduced. The experiment relies on the interaction between the CH bond vector dipole and the (31)P chemical shift anisotropy (CSA), which affects the relaxation of the (13)C,(31)P double- and zero-quantum coherence and thus the intensity of the detectable magnetization. With the new pulse sequence, five different cross-correlated relaxation rates along the phosphodiester backbone can be measured in a quantitative manner, allowing projection-angle and torsion-angle restraints for the two backbone angles alpha and zeta to be extracted. Two versions of the pulse sequence optimized for the CH and CH(2) groups are introduced and demonstrated for a 14-mer cUUCGg tetraloop RNA model system and for a 27-mer RNA with a previously unknown structure. The restraints were incorporated into the calculation of a very high resolution structure of the RNA model system (Nozinovic, S.; et al. Nucleic Acids Res. 2010, 38, 683). Comparison with the X-ray structure of the cUUCGg tetraloop confirmed the high quality of the data, suggesting that the method can significantly improve the quality of RNA structure determination.

High-resolution NMR structure of an RNA model system: the 14-mer cUUCGg tetraloop hairpin RNA.

We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 A) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2'-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined.

RNA phosphodiester backbone dynamics of a perdeuterated cUUCGg tetraloop RNA from phosphorus-31 NMR relaxation analysis.

We have analyzed the relaxation properties of all (31)P nuclei in an RNA cUUCGg tetraloop model hairpin at proton magnetic field strengths of 300, 600 and 900 MHz in solution. Significant H, P dipolar contributions to R (1) and R (2) relaxation are observed in a protonated RNA sample at 600 MHz. These contributions can be suppressed using a perdeuterated RNA sample. In order to interpret the (31)P relaxation data (R (1), R (2)), we measured the (31)P chemical shift anisotropy (CSA) by solid-state NMR spectroscopy under various salt and hydration conditions. A value of 178.5 ppm for the (31)P CSA in the static state (S (2) = 1) could be determined. In order to obtain information about fast time scale dynamics we performed a modelfree analysis on the basis of our relaxation data. The results show that subnanosecond dynamics detected around the phosphodiester backbone are more pronounced than the dynamics detected for the ribofuranosyl and nucleobase moieties of the individual nucleotides (Duchardt and Schwalbe, J Biomol NMR 32:295-308, 2005; Ferner et al., Nucleic Acids Res 36:1928-1940, 2008). Furthermore, the dynamics of the individual phosphate groups seem to be correlated to the 5' neighbouring nucleobases.