The structure of the Thermococcus gammatolerans McrB N-terminal domain reveals a new mode of substrate recognition and specificity among McrB homologs.

McrBC is a two-component, modification-dependent restriction system that cleaves foreign DNA-containing methylated cytosines. Previous crystallographic studies have shown that Escherichia coli McrB uses a base-flipping mechanism to recognize these modified substrates with high affinity. The side chains stabilizing both the flipped base and the distorted duplex are poorly conserved among McrB homologs, suggesting that other mechanisms may exist for binding modified DNA. Here we present the structures of the Thermococcus gammatolerans McrB DNA-binding domain (TgΔ185) both alone and in complex with a methylated DNA substrate at 1.68 and 2.27 Å resolution, respectively. The structures reveal that TgΔ185 consists of a YT521-B homology (YTH) domain, which is commonly found in eukaryotic proteins that bind methylated RNA and is structurally unrelated to the E. coli McrB DNA-binding domain. Structural superposition and co-crystallization further show that TgΔ185 shares a conserved aromatic cage with other YTH domains, which forms the binding pocket for a flipped-out base. Mutational analysis of this aromatic cage supports its role in conferring specificity for the methylated adenines, whereas an extended basic surface present in TgΔ185 facilitates its preferential binding to duplex DNA rather than RNA. Together, these findings establish a new binding mode and specificity among McrB homologs and expand the biological roles of YTH domains.

Zerovalent iron-sand filtration can reduce the concentration of multiple antimicrobials in conventionally treated reclaimed water.

Irrigation with reclaimed water is increasing in areas that lack access to, and infrastructure for, high-level treatment and distribution. Antimicrobial residues are known to persist in conventionally treated reclaimed water, necessitating the investigation of reuse site-based mitigation options to further reduce these contaminants. We examined the effectiveness of a 50:50 volume/volume, particle matched, micro-scale zerovalent iron (ZVI)-sand filter in reducing concentrations of mixtures of antimicrobials present in pH-unadjusted conventionally treated reclaimed water. Twelve antimicrobials (azithromycin, ciprofloxacin, erythromycin, linezolid, oxacillin, oxolinic acid, penicillin G, pipemidic acid, sulfamethoxazole, triclocarban, tetracycline and vancomycin) were quantified using high performance-liquid chromatography-tandem mass spectrometry in reclaimed water, and ZVI-sand filtered reclaimed water, in a two-month long greenhouse-based experiment. Data were analyzed using a non-parametric rank-based approach. ZVI-sand filtration significantly reduced concentrations of azithromycin, ciprofloxacin, oxolinic acid, penicillin G, sulfamethoxazole, linezolid, pipemidic acid and vancomycin. Azithromycin, the antimicrobial with the highest median concentration (320 ng/L), was reduced to below the limit of detection after ZVI-sand filtration. Inorganic element (antimony, beryllium, cadmium, chromium, iron, lead, selenium and thallium) and water quality (free and total chlorine, nitrates, nitrites, pH and total dissolved solids) analyses showed that ZVI-sand filtered reclaimed water quality (nitrate, salinity, and inorganic elements) met the recommended guidelines for agricultural irrigation with reclaimed water. Based on our initial results, ZVI-sand filtration may be a promising basis for a point-of-use filtration system for reclaimed water irrigation on small-scale farms.