Biology subjects

Schaerer, M. R.

Publications and source records attributed to Schaerer, M. R..

3 recordsLinked to original sources

bioRxiv2024

Machine learning reveals signatures of promiscuous microbial amidases for micropollutant biotransformations

Organic micropollutants - including pharmaceuticals, personal care products, pesticides and food additives - are prevalent in the environment and have unknown and potentially toxic effects. Humans are a direct source of micropollutants as the majority of pharmaceuticals are primarily excreted through urine. Urine contains its own microbiota with the potential to catalyze micropollutant biotransformations. Amidase signature (AS) enzymes are known for their promiscuous activity in micropollutant biotransformations, but the potential for AS enzymes from the urinary microbiota to transform micropollutants is not known. Moreover, characterization of AS enzymes to identify key chemical and enzymatic features predictive of biotransformation profiles is critical for developing benign-by-design chemicals and micropollutant removal strategies. In this study, we biochemically characterized a new AS enzyme with arylamidase activity from a urine isolate, Lacticaseibacillus rhamnosus, and demonstrated its capability to hydrolyze pharmaceuticals and other micropollutants. To uncover the signatures of AS enzyme-substrate specificity, we then designed a targeted enzyme library consisting of 40 arylamidase homologs from diverse urine isolates and tested it against 17 structurally diverse compounds. We found that 16 out of the 40 enzymes showed activity on at least one substrate and exhibited diverse substrate specificities, with the most promiscuous enzymes active on nine different substrates. Using an interpretable gradient boosting machine learning model, we identified chemical and amino acid features predictive of arylamidase biotransformations. Key chemical features from our substrates included the molecular weight of the amide carbonyl substituent and the number of charges in the molecule. Important amino acid features were found to be located on the protein surface and four predictive residues were located in close proximity of the substrate tunnel entrance. Overall, this work highlights the understudied role of urine-derived microbial arylamidases and contributes to enzyme sequence-structure-substrate-based predictions of micropollutant biotransformations.

biochemistry↗

bioRxiv2023

Disentangling abiotic and biotic effects of treated wastewater on stream biofilm resistomes enables the discovery of a new planctomycete beta-lactamase

BackgroundAntibiotic resistance, which is mediated by environmental reservoirs, poses a threat to human and animal health. Aquatic biofilms impacted by treated wastewater (WW) are known environmental reservoirs for antibiotic resistance, however the specific influence of biotic factors and abiotic factors from WW on the abundance of antibiotic resistance genes (ARGs) within aquatic biofilms remains unclear. Additionally, experimental evidence is limited as to whether genes with low sequence similarity to reference ARGs actually encode for functional ARGs, particularly within complex aquatic microbial communities. ResultsTo disentangle the effects of abiotic and biotic factors on ARG abundances, natural biofilms were previously grown in flume systems with different proportions of stream water and either ultrafiltered or nonultrafiltered WW. In this study, we conducted deep shotgun metagenomic sequencing of 75 biofilm, stream, and WW samples from these flume systems and compared the taxonomic and functional microbiome and resistome composition. Statistical analysis revealed an alignment of the resistome and microbiome composition and a significant association with experimental treatment. Several ARG classes exhibited an increase in metagenomic abundances in biofilms grown with increasing percentages of nonultrafiltered WW. In contrast, sulfonamide and BEL family beta-lactamase ARGs showed greater abundances in biofilms grown in ultrafiltered WW compared to nonultrafiltered WW. Overall, our results pointed toward the dominance of biotic factors over abiotic factors in determining ARG abundances in WW-impacted stream biofilms and suggested gene family-specific mechanisms for ARGs which exhibited divergent abundance patterns. To investigate one of these specific ARG families experimentally, we biochemically characterized a new beta-lactamase from the Planctomycetota (Phycisphaeraceae). This beta-lactamase displayed activity in the cleavage of cephalosporin analog despite sharing low sequence identity with known ARGs. ConclusionsThis discovery of a functional planctomycete beta-lactamase ARG is noteworthy, not only because it was the first beta-lactamase to be biochemically characterized from this phylum, but also because it was not detected by standard homology-based ARG tools. In summary, this study conducted metagenomic analysis on the relative importance of biotic and abiotic factors in the context of WW discharge and their impact on both known and new ARGs in aquatic biofilms.

microbiology↗

bioRxiv2023

Substrate promiscuity of xenobiotic-transforming hydrolases from stream biofilms impacted by treated wastewater

Organic contaminants enter aquatic ecosystems from various sources, including wastewater treatment plant effluent. Freshwater biofilms play a major role in the removal of organic contaminants from receiving water bodies, but knowledge of the molecular mechanisms driving contaminant biotransformations in complex stream biofilm (periphyton) communities remains limited. Previously, we demonstrated that biofilms in experimental flume systems grown at higher ratios of treated wastewater (WW) to stream water displayed an increased biotransformation potential for a number of organic contaminants. We identified a positive correlation between WW percentage and biofilm biotransformation rates for the widely-used insect repellent, N,N-diethyl-meta-toluamide (DEET). Here, we conducted deep shotgun sequencing of flume biofilms and identified a positive correlation between WW percentage and metagenomic read abundances of DEET hydrolase (DH) homologs. To test the causality of this association, we constructed a targeted metagenomic library of DH homologs from flume biofilms. We screened our complete metagenomic library for activity with four different substrates and a subset thereof with 183 WW-related organic compounds. The majority of active hydrolases in our library preferred aliphatic and aromatic ester substrates while, remarkably, only a single reference enzyme was capable of DEET hydrolysis. Of the 626 total enzyme-substrate combinations tested, approximately 5% were active enzyme-substrate pairs. Metagenomic DH family homologs revealed a broad substrate promiscuity spanning 22 different compounds when summed across all enzymes tested. We biochemically characterized the most promiscuous and active enzymes identified based on metagenomic analysis from uncultivated Rhodospirillaceae and Planctomycetaceae. In addition to characterizing new DH family enzymes, we exemplified a framework for linking metagenome-guided hypothesis generation with experimental validation. Overall, this study expands the scope of known enzymatic contaminant biotransformations for metagenomic hydrolases from WW-receiving stream biofilm communities. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/559296v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1b5cbaforg.highwire.dtl.DTLVardef@4f73f7org.highwire.dtl.DTLVardef@467d16org.highwire.dtl.DTLVardef@1be598d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMetagenomic DEET hydrolase abundances higher in biofilms grown in treated wastewater. C_LIO_LIEleven out of 64 metagenomic hydrolases tested exhibited hydrolysis activity. C_LIO_LIRelated enzymes in a single family of DEET hydrolases biotransform 20+ contaminants. C_LIO_LIReference DEET hydrolase shows a substrate preference for benzamide moieties. C_LIO_LI True DEET hydrolases are in low abundance even in biofilms that degrade DEET. C_LI

biochemistry↗