Structural insight into the functional mechanism of Nep1/Emg1 N1-specific pseudouridine methyltransferase in ribosome biogenesis

Nucleolar Essential Protein 1 (Nep1) is required for small subunit (SSU) ribosomal RNA (rRNA) maturation and is mutated in Bowen–Conradi Syndrome. Although yeast (Saccharomyces cerevisiae) Nep1 interacts with a consensus sequence found in three regions of SSU rRNA, the molecular details of the interaction are unknown. Nep1 is a SPOUT RNA methyltransferase, and can catalyze methylation at the N1 of pseudouridine. Nep1 is also involved in assembly of Rps19, an SSU ribosomal protein. Mutations in Nep1 that result in decreased methyl donor binding do not result in lethality, suggesting that enzymatic activity may not be required for function, and RNA binding may play a more important role. To study these interactions, the crystal structures of the scNep1 dimer and its complexes with RNA were determined. The results demonstrate that Nep1 recognizes its RNA site via base-specific interactions and stabilizes a stem-loop in the bound RNA. Furthermore, the RNA structure observed contradicts the predicted structures of the Nep1-binding sites within mature rRNA, suggesting that the Nep1 changes rRNA structure upon binding. Finally, a uridine base is bound in the active site of Nep1, positioned for a methyltransfer at the C5 position, supporting its role as an N1-specific pseudouridine methyltransferase

Methylation QTLs in the developing brain and their enrichment in schizophrenia risk loci

We characterized DNA methylation quantitative trait loci (mQTLs) in a large collection (n = 166) of human fetal brain samples spanning 56-166 d post-conception, identifying >16,000 fetal brain mQTLs. Fetal brain mQTLs were primarily cis-acting, enriched in regulatory chromatin domains and transcription factor binding sites, and showed substantial overlap with genetic variants that were also associated with gene expression in the brain. Using tissue from three distinct regions of the adult brain (prefrontal cortex, striatum and cerebellum), we found that most fetal brain mQTLs were developmentally stable, although a subset was characterized by fetal-specific effects. Fetal brain mQTLs were enriched amongst risk loci identified in a recent large-scale genome-wide association study (GWAS) of schizophrenia, a severe psychiatric disorder with a hypothesized neurodevelopmental component. Finally, we found that mQTLs can be used to refine GWAS loci through the identification of discrete sites of variable fetal brain methylation associated with schizophrenia risk variants

The efficacy of different antimicrobial metals at preventing the formation of, and eradicating bacterial biofilms of pathogenic indicator strains.

The emergence of multidrug resistant pathogens and the prevalence of biofilm-related infections have generated a demand for alternative antimicrobial therapies. Metals have not been explored in adequate detail for their capacity to combat infectious disease. Metal compounds can now be found in textiles, medical devices, and disinfectants – yet, we know little about their efficacy against specific pathogens. To help fill this knowledge gap, we report on the antimicrobial and antibiofilm activity of seven metals; silver, copper, titanium, gallium, nickel, aluminum and zinc against three bacterial strains, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. In order to evaluate the capacity of metal ions to prevent the growth of, and eradicate biofilms and planktonic cells, bacterial cultures were inoculated in the Calgary Biofilm Device (MBEC™) in the presence the metal salts. Copper, gallium, and titanium were capable of preventing planktonic and biofilm growth, and eradicating established biofilms of all tested strains. Further, we observed that the efficacies of the other tested metal salts displayed variable efficacy against the tested strains. Further, contrary to the enhanced resistance anticipated from bacterial biofilms, particular metal salts were observed to be more effective against biofilm communities versus planktonic cells. In this study, we have demonstrated that the identity of the bacterial strain must be considered prior to treatment with a particular metal ion. Consequently, as the use of metal ions as antimicrobial agents to fight multidrug resistant and biofilm related infections increases, we must aim for more selective deployment in a given infectious setting.NSERC, AIHS,Ye

Evaluating the Metal Tolerance Capacity of Microbial Communities Isolated from Alberta Oil Sands Process Water.

Anthropogenic activities have resulted in the intensified use of water resources. For example, open pit bitumen extraction by Canada's oil sands operations uses an estimated volume of three barrels of water for every barrel of oil produced. The waste tailings-oil sands process water (OSPW)-are stored in holding ponds, and present an environmental concern as they are comprised of residual hydrocarbons and metals. Following the hypothesis that endogenous OSPW microbial communities have an enhanced tolerance to heavy metals, we tested the capacity of planktonic and biofilm populations from OSPW to withstand metal ion challenges, using Cupriavidus metallidurans, a known metal-resistant organism, for comparison. The toxicity of the metals toward biofilm and planktonic bacterial populations was determined by measuring the minimum biofilm inhibitory concentrations (MBICs) and planktonic minimum inhibitory concentrations (MICs) using the MBEC ™ assay. We observed that the OSPW community and C. metallidurans had similar tolerances to 22 different metals. While thiophillic elements (Te, Ag, Cd, Ni) were found to be most toxic, the OSPW consortia demonstrated higher tolerance to metals reported in tailings ponds (Al, Fe, Mo, Pb). Metal toxicity correlated with a number of physicochemical characteristics of the metals. Parameters reflecting metal-ligand affinities showed fewer and weaker correlations for the community compared to C. metallidurans, suggesting that the OSPW consortia may have developed tolerance mechanisms toward metals present in their environment

A novel approach for harnessing biofilm communities in moving bed biofilm reactors for industrial wastewater treatment

Moving bed biofilm reactors (MBBRs) are an effective biotechnology for treating industrial wastewater. Biomass retention on moving bed biofilm reactor (MBBR) carriers (biofilm support materials), allows for the ease-of-operation and high treatment capacity of MBBR systems. Optimization of MBBR systems has largely focused on aspects of carrier design, while little attention has been paid to enhancing strategies for harnessing microbial biomass. Previously, our research group demonstrated that mixed-species biofilms can be harvested from an industrial wastewater inoculum [oil sands process water (OSPW)] using the Calgary Biofilm Device (CBD). Moreover, the resultant biofilm communities had the capacity to degrade organic toxins (naphthenic acids—NAs) that are found in OSPW. Therefore, we hypothesized that harnessing microbial communities from industrial wastewater, as biofilms, on MBBR carriers may be an effective method to bioremediate industrial wastewater.Here, we detail our methodology adapting the workflow employed for using the CBD, to generate inoculant carriers to seed an MBBR.In this study, OSPW-derived biofilm communities were successfully grown, and their efficacy evaluated, on commercially available MBBR carriers affixed within a modified CBD system. The resultant biofilms demonstrated the capacity to transfer biomass to recipient carriers within a scaled MBBR. Moreover, MBBR systems inoculated in this manner were fully active 2 days post-inoculation, and readily degraded a select population of NAs. Together, these findings suggest that harnessing microbial communities on carriers affixed within a modified CBD system may represent a facile and rapid method for obtaining functional inoculants for use in wastewater MBBR treatment systems

Planktonic minimal inhibitory concentrations plotted against pK_sp.

<p>Significant correlation is evident with the OSPW community (A), and absent with <i>C</i>. <i>metallidurans</i> (B). Dashed lines show 95% confidence bands.</p

Linear regression analysis of physicochemical parameters with metal toxicity values for OSPW cultures and <i>C</i>. <i>metallidurans</i>, represented as minimum inhibitory concentrations (MIC) and minimum biofilm inhibitory concentrations (MBIC).

<p>Linear regression analysis of physicochemical parameters with metal toxicity values for OSPW cultures and <i>C</i>. <i>metallidurans</i>, represented as minimum inhibitory concentrations (MIC) and minimum biofilm inhibitory concentrations (MBIC).</p

Linear regression analysis of MBIC values plotted against physicochemical parameters for OSPW consortia.

<p>MBIC correlations with physicochemical parameters illustrate a typical metal susceptibility profile of the OSPW community. Parameters include: metal-sulfide solubility product (pK_sp), electronegativity (X_m), standard reduction-oxidation potentials (ΔE₀), Pearson’s softness index (σ_p), first ionization energy (<i>I</i>_<i>1</i>), and first hydrolysis constant (|Log K_OH|). Trend lines and 95% confidence bands (dashed lines) shown on linear regressions that correlate with significance.</p

Silver oxynitrate – an efficacious compound for the prevention and eradication of dual-species biofilms

<p>Preventing and eradicating biofilms remains a challenge in clinical and industrial settings. Recently, the present authors demonstrated that silver oxynitrate (Ag₇NO₁₁) prevented and eradicated single-species planktonic and biofilm populations of numerous microbes at lower concentrations than other silver (Ag) compounds. Here, the antimicrobial and anti-biofilm efficacy of Ag₇NO₁₁ is elaborated by testing its <i>in vitro</i> activity against combinations of dual-species, planktonic and biofilm populations of <i>Escherichia coli</i>, <i>Staphylococcus aureus</i> and <i>Pseudomonas aeruginosa</i>. As further evidence emerges that multispecies bacterial communities are more common in the environment than their single-species counterparts, this study reinforces the diverse applicability of the minimal biofilm eradication concentration (MBEC™) assay for testing antimicrobial compounds against biofilms. Furthermore, this study demonstrated that Ag₇NO₁₁ had enhanced antimicrobial and anti-biofilm activity compared to copper sulfate (CuSO₄) and silver nitrate (AgNO₃) against the tested bacterial species.</p