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Barren, metal-contaminated soils lack plants and root exudate inputs, exhibit low microbial abundance and functioning, and often require soil revitalization to revegetate. While the effects of simulated root exudates (SRE) have been investigated in uncontaminated, vegetated soils, their potential for remediating post-industrial barren, contaminated soils has not been examined or leveraged. We asked whether priming brownfield soils with a laboratory-prepared SRE solution stimulates native soil microbial metabolism and functioning and how long the effects last. Moreover, we compared a cost-effective single SRE addition to repeated SRE additions. We collected soils from a metal-contaminated, abandoned industrial rail yard (barren and vegetated sites) and a vegetated agricultural reference site, established microcosms, and treated the soils with either a single or repeated SRE addition/s. By day 30, SRE-enriched barren, brownfield soils showed significantly higher soil respiration rates than the untreated control soils. Phosphatase activities were significantly higher even 210 days after a single SRE addition. Plants were introduced 282 days after the single SRE addition. The average shoot height (16 ± 0.3 cm) and total plant biomass (0.5 ± 0.02 g) of plants grown in single addition SRE enriched barren soil were significantly higher than the controls (9 ± 0.9 cm and 0.3 ± 0.02 g, respectively). The increased soil microbial functioning and productivity indicate that a single SRE application holds promise as a field-ready technology to revitalize barren, poorly functioning brownfield soils. SRE application may also be a pragmatic and innovative approach to enable successful phytoremediation and re-greening of industrial barrens. 

Many antibacterial and antiparasitic drugs work by competitively inhibiting dihydrofolate reductase (DHFR), a vital enzyme in folate metabolism. The interactions between inhibitors and DHFR active site residues are known in many homologs but the contributions from distal residues are less understood. Identifying distal residues that aid in inhibitor binding can improve targeted drug development programs by accounting for distant influences that may be less conserved and subject to frequent resistance causing mutations. Previously, a novel, homologybased, computational approach that mines ligand inhibition data was used to predict residues involved in inhibitor selectivity in the DHFR family. Expectedly, some inhibitor selectivity determining residue positions were predicted to lie in the active site and coincide with experimentally known inhibitor selectivity determining positions. However, other residues that group spatially in clusters distal to the active site have not been previously investigated. In this study, the effect of introducing amino acid substitutions at one of these predicted clusters (His38-Ala39-Ile40) on the inhibitor selectivity profile in Bacillus stearothermophilus dihydrofolate reductase (Bs DHFR) was investigated. Mutations were introduced into these cluster positions to change sidechain chemistry and size. We determined kcat and KM values and measured KD values at equilibrium for two competitive DHFR inhibitors, trimethoprim (TMP) and pyrimethamine (PYR). Mutations in the His38-Ala39-Ile40 cluster significantly impacted inhibitor binding and TMP/PYR selectivity - seven out of nine mutations resulted in tighter binding to PYR when compared to TMP. These data suggest that the His38-Ala39-Ile40 cluster is a distal inhibitor selectivity determining region that favors PYR binding in Bs DHFR and, possibly, throughout the DHFR family.