Biology subjects

Demirer, K.

Publications and source records attributed to Demirer, K..

4 recordsLinked to original sources

bioRxiv2026

Genome-wide fitness profiling reveals flagellar rotation as an energetic liability during anaerobic maintenance in Pseudomonas aeruginosa

Bacteria in nature and disease frequently spend time in non-growing states, yet the genes that allow cells to survive without growing remain poorly understood. Using Pseudomonas aeruginosa strain PA14 as a model system to study non-growth powered by anaerobic phenazine cycling, we employed randomly barcoded transposon-insertion sequencing (RB-TnSeq) to identify the genes required for this maintenance state using a platform that sustains anaerobic survival via continuous phenazine reoxidation. We found 167 genes whose disruption altered survival, including genes involved in transcription, translation, protein quality control, and cell envelope maintenance. Comparing our results to published TnSeq data from other forms of energy-limited growth arrest (conditions where electron acceptor or carbon availability constrains energy conservation below that required for cell growth/division) in PA14 revealed that while a subset of genes are fitness determinants across distinct growth-arrested states, most are condition-specific. Notably, genes involved in flagellar regulation and assembly were broadly detrimental to survival under this maintenance condition. Leveraging a high-throughput electrochemical system that allows for quantitative and mechanistic dissection of the phenazine cycling-dependent maintenance state, we found that flagellar abundance influences cells survival, metabolic rate, and ATP levels. Moreover, removing the flagellar stator proteins MotAC, which are required for flagellar rotation but not assembly, reversed these defects, indicating that the energetic cost of flagella comes from their rotation rather than construction under these conditions. These results show that in a low-powered maintenance state, limiting energy-dissipating processes, such as proton-motive force loss through flagellar rotation, supports cell survival.

microbiology↗

bioRxiv2026

Structural basis of metalloid transport by the arsenite efflux pump ArsB

Bacteria resist toxic arsenite (AsIII) in their environments by actively pumping the metalloid out of the cell via efflux pumps such as ArsB. However, the mechanism of extrusion remains poorly understood, which hinders the development of engineered bioremediation strategies. We report high-resolution cryo-EM structures of ArsB from the arsenic-tolerant bacterium Leptospirillum ferriphilum. ArsB adopts an inverted two-fold repeat architecture resembling that of other ion transporter (IT) superfamily proteins. Structures determined in the presence of AsIII and antimonite (SbIII) reveal that the metalloid substrates interact with polar residues at the core of the transmembrane domain primarily via hydrogen bonding. Mutagenesis and in vivo functional assays support these interactions. Our ArsB structures represent an inward-facing conformation, where the metalloid-binding site is exposed to the cytoplasm, suitable for metalloid capture. Furthermore, we demonstrate that AsIII resistance conferred by ArsB varies with external pH, supporting that ArsB is a proton (H+)-coupled secondary transporter. Mutagenesis, in vivo functional assays, and pKa estimation imply that conserved aspartate residues near the metalloid-binding site likely mediate the H+-coupling mechanism. Our findings provide structural insights into metalloid recognition and H+/metalloid antiport in ArsB, laying a foundation for further elucidation of the molecular basis of toxic metalloid detoxification in bacteria.

biochemistry↗

bioRxiv2023

Transport of metformin metabolites by guanidinium exporters of the Small Multidrug Resistance family

Proteins from the Small Multidrug Resistance (SMR) family are frequently associated with horizontally transferred multidrug resistance gene arrays found in bacteria from wastewater and the human-adjacent biosphere. Recent studies suggest that a subset of SMR transporters might participate in metabolism of the common pharmaceutical metformin by bacterial consortia. Here, we show that both genomic and plasmid-associated transporters of the SMRGdx functional subtype export byproducts of microbial metformin metabolism, with particularly high export efficiency for guanylurea. We use solid supported membrane electrophysiology to evaluate the transport kinetics for guanylurea and native substrate guanidinium by four representative SMRGdx homologues. Using an internal reference to normalize independent electrophysiology experiments, we show that transport rates are comparable for genomic and plasmid-associated SMRGdx homologues, and using a proteoliposome-based transport assay, we show that 2 proton:1 substrate transport stoichiometry is maintained. Additional characterization of guanidinium and guanylurea export properties focuses on the structurally characterized homologue, Gdx-Clo, for which we examined the pH dependence and thermodynamics of substrate binding and solved an x-ray crystal structure with guanylurea bound. Together, these experiments contribute in two main ways. By providing the first detailed kinetic examination of the structurally characterized SMRGdx homologue Gdx-Clo, they provide a functional framework that will inform future mechanistic studies of this model transport protein. Second, this study casts light on a potential role for SMRGdx transporters in microbial handling of metformin and its microbial metabolic byproducts, providing insight into how native transport physiologies are co-opted to contend with new selective pressures. SummaryUsing solid supported membrane electrophysiology, structural biology, and binding assays, we characterize binding and transport of metformin metabolites by bacterial SMR transporters, including proteins associated with horizontal gene transfer in wastewater bacteria that degrade metformin.

biophysics↗

bioRxiv2020

Lanthanide-dependent alcohol dehydrogenases require an essential aspartate residue for metal coordination and function

The presence of lanthanide elements (Ln3+) and pyrroloquinoline quinone (PQQ) containing cofactors in XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs) has expanded the list of biological elements and opened novel areas of metabolism and ecology. Other MDHs known as MxaFIs are related in sequence and structure to these proteins, yet they instead possess a Ca2+-PQQ cofactor. An important missing piece of the Ln3+ puzzle is defining what protein features distinguish enzymes using Ln3+-PQQ cofactors from those that do not. In this study, we investigated the functional importance of a proposed lanthanide-coordinating aspartate using XoxF1 MDH from the model methylotrophic bacterium Methylorubrum extorquens AM1. We report two crystal structures of XoxF1, one containing PQQ and the other free of this organic molecule, both with La3+ bound in the active site region and coordinated by Asp320. Using constructs to produce either recombinant XoxF1 or its D320A variant, we show Asp320 is needed for in vivo catalytic function, in vitro activity of purified enzyme, and coordination of La3+. XoxF1 and XoxF1 D320A, when produced in the absence of La3+, coordinate Ca2+, but exhibit little or no catalytic activity. In addition, we generated the parallel substitution to produce ExaF D319S, and showed the enzyme loses the capacity for efficient ethanol oxidation with La3+. These results provide empirical evidence of an essential Ln3+-coordinating aspartate for the function of XoxF MDHs and ExaF EDHs; thus, supporting the suggestion that sequences of these enzymes, and the genes that encode them, are markers for Ln3+ metabolism.

biochemistry↗