Biology subjects

Manefield, M. J.

Publications and source records attributed to Manefield, M. J..

3 recordsLinked to original sources

bioRxiv2026

Selective Hydrolytic Defluorination of Branched Perfluorooctanoic Acid Isomers by a Haloacid Dehalogenase

Per- and polyfluoroalkyl substances (PFAS) are highly resistant to enzymatic C-F bond cleavage, and hydrolytic defluorination of long-chain PFAS has rarely been demonstrated. Here, we report selective hydrolytic defluorination of branched perfluorooctanoic acid (PFOA) isomers by a haloacid dehalogenase (4A) from Delftia acidovorans strain D4B. A fluoride-specific riboswitch biosensor was used for initial substrate screening, followed by scaled-up assays in which fluoride release was quantified using a fluoride ion-selective electrode. Defluorination products were subsequently identified by liquid chromatography-mass spectrometry (LC-MS). Although purified 4A (10 M) readily catalyzed hydrolytic defluorination of fluoroacetic acid, incubation of PFOA (0.5 mM) with purified 4A resulted in a statistically significant increase in fluoride release at elevated enzyme loading (500 M). High-resolution LC-MS/MS analysis revealed that defluorination products originated from minor branched PFOA isomers rather than linear PFOA. Molecular docking analyses supported catalytically plausible binding geometries for branched PFOA isomers, positioning the substrate -carbon within [~]4 [A] of the catalytic aspartate residue. These findings demonstrate previously unrecognized hydrolytic reactivity of a haloacid dehalogenase toward branched PFAS isomers and expand the known catalytic scope of the haloacid dehalogenase family. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/719434v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@d56316org.highwire.dtl.DTLVardef@15c7961org.highwire.dtl.DTLVardef@24de56org.highwire.dtl.DTLVardef@80f19d_HPS_FORMAT_FIGEXP M_FIG C_FIG SYNOPSISEnzymatic defluorination of PFAS is rarely observed in environmental systems. This study identifies hydrolytic defluorination of branched PFOA isomers, improving understanding of PFAS defluorination at the enzyme level.

synthetic biology↗

bioRxiv2022

Evidence for a putative isoprene reductase in Acetobacterium wieringae

Recent discoveries of isoprene-metabolizing microorganisms suggest they might play an important role in the global isoprene budget. Under anoxic conditions, isoprene can be used as an electron acceptor and is reduced to methylbutene. This study describes the proteogenomic profiling of an isoprene-reducing bacterial culture to identify organisms and genes responsible for the isoprene hydrogenation reaction. A metagenome assembled genome (MAG) of the most abundant (89% rel. abundance) lineage in the enrichment, Acetobacterium wieringae, was obtained. Comparative proteogenomics and RT-PCR identified a putative five-gene operon from the A. wieringae MAG upregulated during isoprene reduction. The operon encodes a putative oxidoreductase, three pleiotropic nickel chaperones (2 x HypA-like, HypB-like) and one 4Fe-4S ferredoxin. The oxidoreductase is proposed as the putative isoprene reductase with a binding site for NADH, FAD and two pairs of [4Fe-4S]-clusters. Other known Acetobacterium strains do not encode the isoprene-regulated operon but encode, like many other bacteria, a homolog of the putative isoprene reductase ([~]47-49% amino acid sequence identity). Uncharacterized homologs of the putative isoprene reductase are observed across the Firmicutes, Spirochaetes, Tenericutes, Actinobacteria, Chloroflexi, Bacteroidetes and Proteobacteria, suggesting the ability of biohydrogenation of unfunctionalized conjugated doubled bonds in other unsaturated hydrocarbons. ImportanceIsoprene was recently shown to act as an electron acceptor for a homoacetogenic bacterium. The focus of this study is the molecular basis for isoprene reduction. By comparing a genome from our isoprene reducing enrichment culture, dominated by Acetobacterium wieringae, with genomes of other Acetobacterium lineages that do not reduce isoprene, we shortlisted candidate genes for isoprene reduction. Using comparative proteogenomics and reverse transcription PCR we have identified a putative five-gene operon encoding an oxidoreductase referred to as putative isoprene reductase.

microbiology↗

bioRxiv2020

A novel, dichloromethane-fermenting bacterium in the Peptococcaceae family, 'Candidatus Formamonas warabiya', gen. nov. sp. nov.

Dichloromethane (DCM; CH2Cl2) is a toxic groundwater pollutant that also has a detrimental effect on atmospheric ozone levels. As a dense non-aqueous phase liquid, DCM migrates vertically through groundwater to low redox zones, yet information on anaerobic microbial DCM transformation remains scarce due to a lack of cultured organisms. We report here the characterisation of strain DCMF, the dominant organism in an anaerobic enrichment culture (DFE) that is capable of fermenting DCM to the environmentally benign product acetate. Stable carbon isotope experiments demonstrated that the organism assimilated carbon from DCM and bicarbonate via the Wood-Ljungdahl pathway. Strain DCMF is the first anaerobic DCM-degrading bacterium also shown to metabolise non-chlorinated substrates. It appears to be a methylotroph utilising the Wood-Ljungdahl pathway for metabolism of methyl groups from methanol, choline, and glycine betaine, which has implications for the flux of climate-active compounds from subsurface environments. Community profiling and enrichment of the cohabiting taxa in culture DFE to the exclusion of strain DCMF suggest that it is the sole organism in this culture responsible for substrate metabolism, while the cohabitants persist via necromass recycling. Genomic and physiological evidence support placement of strain DCMF in a novel genus, Candidatus Formamonas warabiya.

microbiology↗