Extracellular Electron Transfer: Respiratory or Nutrient Homeostasis?

Exoelectrogens are able to transfer electrons extracellularly, enabling them to respire on insoluble terminal electron acceptors. Extensively studied exoelectrogens, such as Geobacter sulfurreducens and Shewanella oneidensis, are Gram negative. More recently, it has been reported that Gram-positive bacteria, such as Listeria monocytogenes and Enterococcus faecalis, also exhibit the ability to transfer electrons extracellularly, although it is still unclear whether this has a function in respiration or in redox control of the environment, for instance, by reducing ferric iron for iron uptake. In this issue of Journal of Bacteriology, Hederstedt and colleagues report on experiments that directly compare extracellular electron transfer (EET) pathways for ferric iron reduction and respiration and find a clear difference (L. Hederstedt, L. Gorton, and G. Pankratova, J Bacteriol 202:e00725-19, 2020, https://doi.org/10.1128/JB.00725-19), providing further insights and new questions into the function and metabolic pathways of EET in Gram-positive bacteria

Shewanella oneidensis MR-1 electron acceptor taxis and the perception of electrodes poised at oxidative potentials

Shewanella oneidensis MR-1 is a facultative anaerobe, capable of respiring on an extraordinarily large and diverse array of both intra- and extracellular terminal electron acceptors, including insoluble metal oxides and electrodes. The ability to perform extracellular electron transfer has sparked great interest over the last three decades and MR-1 has become both a model organism for fundamental research into extracellular electron transfer and a candidate microbe for microbial electrochemical systems, including microbial fuel cells. A pre-requisite for colonisation and biofilm formation on electrodes is the migration of bacteria towards the electrode. Here, we review current understanding in the steps involved in MR-1 migration towards insoluble electron acceptors and electrodes. The main experimental techniques used to evaluate taxis are summarised and different mechanisms proposed for MR-1 taxis are contrasted, in particular chemotaxis versus energy taxis

Engineering Protein Switches for Rapid Diagnostic Tests

Biological signaling pathways are underpinned by protein switches that sense and respond to molecular inputs. Inspired by nature, engineered protein switches have been designed to directly transduce analyte binding into a quantitative signal in a simple, wash-free, homogeneous assay format. As such, they offer great potential to underpin point-of-need diagnostics that are needed across broad sectors to improve access, costs, and speed compared to laboratory assays. Despite this, protein switch assays are not yet in routine diagnostic use, and a number of barriers to uptake must be overcome to realize this potential. Here, we review the opportunities and challenges in engineering protein switches for rapid diagnostic tests. We evaluate how their design, comprising a recognition element, reporter, and switching mechanism, relates to performance and identify areas for improvement to guide further optimization. Recent modular switches that enable new analytes to be targeted without redesign are crucial to ensure robust and efficient development processes. The importance of translational steps toward practical implementation, including integration into a user-friendly device and thorough assay validation, is also discussed

Tactic Response of Shewanella oneidensis MR-1 toward Insoluble Electron Acceptors

Exoelectrogenic bacteria are defined by their ability to respire on extracellular and insoluble electron acceptors and have applications in bioremediation and microbial electrochemical systems (MESs), while playing important roles in biogeochemical cycling. Shewanella oneidensis MR-1, which has become a model organism for the study of extracellular respiration, is known to display taxis toward insoluble electron acceptors, including electrodes. Multiple mechanisms have been proposed for MR-1’s tactic behavior, and, here, we report on the role of electrochemical potential by video microscopy cell tracking experiments in three-electrode electrochemical cells. MR-1 trajectories were determined using a particle tracking algorithm and validated with Shannon’s entropy method. Tactic response by MR-1 in the electrochemical cell was observed to depend on the applied potential, as indicated by the average velocity and density of motile (>4 µm/s) MR-1 close to the electrode (<50 µm). Tactic behavior was observed at oxidative potentials, with a strong switch between the potentials −0.15 to −0.25 V versus the standard hydrogen electrode (SHE), which coincides with the reduction potential of flavins. The average velocity and density of motile MR-1 close to the electrode increased when riboflavin was added (2 µM), but were completely absent in a ΔmtrC/ΔomcA mutant of MR-1. Besides flavin’s function as an electron mediator to support anaerobic respiration on insoluble electron acceptors, we propose that riboflavin is excreted by MR-1 to sense redox gradients in its environment, aiding taxis toward insoluble electron acceptors, including electrodes in MESs

Hybrid Vesicle Stability under Sterilisation and Preservation Processes Used in the Manufacture of Medicinal Formulations

Sterilisation and preservation of vesicle formulations are important considerations for their viable manufacture for industry applications, particular those intended for medicinal use. Here, we undertake an initial investigation of the stability of hybrid lipid-block copolymer vesicles to common sterilisation and preservation processes, with particular interest in how the block copolymer component might tune vesicle stability. We investigate two sizes of polybutadiene-block-poly(ethylene oxide) polymers (PBd12-PEO11 and PBd22-PEO14) mixed with the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) considering the encapsulation stability of a fluorescent cargo and the colloidal stability of vesicle size distributions. We find that autoclaving and lyophilisation cause complete loss of encapsulation stability under the conditions studied here. Filtering through 200 nm pores appears to be viable for sterilisation for all vesicle compositions with comparatively low release of encapsulated cargo, even for vesicle size distributions which extend beyond the 200 nm filter pore size. Freeze-thaw of vesicles also shows promise for the preservation of hybrid vesicles with high block copolymer content. We discuss the process stability of hybrid vesicles in terms of the complex mechanical interplay between bending resistance, stretching elasticity and lysis strain of these membranes and propose strategies for future work to further enhance the process stability of these vesicle formulations

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis

Electrophysiology Measurements of Metal Transport by MntH2 from Enterococcus faecalis

Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterised using metal-sensitive fluorescent dyes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with an electrophysiology method that is based on the solid-supported membrane technology. E. faecalis MntH2 belongs to the Natural Resistance-Associated Macrophage Protein (Nramp) family of proton-coupled transporters, which transport divalent transition metals and do not transport the earth metals. Electrophysiology confirms transport of Mn(II), Co(II), Zn(II) and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) and Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Fluorescence assays confirm that Ni(II) is transported. The data are discussed with respect to properties and structures of Nramp-type family members and the ability of electrophysiology to measure charge transport and not directly substrate transport

Quantum dot interactions with and toxicity to Shewanella oneidensis MR-1

Combining abiotic photosensitisers such as quantum dots (QDs) with non-photosynthetic bacteria presents an intriguing concept into the design of artificial photosynthetic organisms and solar-driven fuel production. Shewanella oneidensis MR-1 (MR-1) is a versatile bacterium concerning respiration, metabolism and biocatalysis, and is a promising organism for artificial photosynthesis as the bacterium's synthetic and catalytic ability provides a potential system for bacterial biohydrogen production. MR-1's hydrogenases are present in the periplasmatic space. It follows that for photoenergised electrons to reach these enzymes, QDs will need to be able to enter the periplasm, or electrons need to enter the periplasm via the Mtr pathway that is responsible for MR-1's extracellular electron transfer ability. As a step towards this goal, various QDs were tested for their photo-reducing potential, nanotoxicology and further for their interaction with MR-1. CdTe/CdS/TGA, CdTe/CdS/Cysteamine, a commercial, negatively charged CdTe and CuInS2/ZnS/PMAL QDs were examined. The photoreduction potential of the QDs was confirmed by measuring their ability to photoreduce methyl viologen with different sacrificial electron donors. The commercial CdTe and CuInS2/ZnS/PMAL QDs showed no toxicity towards MR-1 as evaluated by a colony-forming units method and a fluorescence viability assay. Only the commercial negatively charged CdTe QDs showed good interaction with MR-1. With transmission electron microscopy, QDs were observed both in the cytoplasm and periplasm. These results inform on the possibilities and bottlenecks when developing bionanotechnological systems for the photosynthetic production of biohydrogen by MR-1

A Systematic Review of the Effect of Therapeutic Drug Monitoring on Patient Health Outcomes during Treatment with Carbapenems

Adjusting dosing regimens based on measurements of carbapenem levels may improve carbapenem exposure in patients. This systematic review aims to describe the effect carbapenem therapeutic drug monitoring (TDM) has on health outcomes, including the emergence of antimicrobial resistance (AMR). Four databases were searched for studies that reported health outcomes following adjustment to dosing regimens, according to measurements of carbapenem concentration. Bias in the studies was assessed with risk of bias analysis tools. Study characteristics and outcomes were tabulated and a narrative synthesis was performed. In total, 2 randomised controlled trials (RCTs), 17 non-randomised studies, and 19 clinical case studies were included. Significant variation in TDM practice was seen; consequently, a meta-analysis was unsuitable. Few studies assessed impacts on AMR. No significant improvement on health outcomes and no detrimental effects of carbapenem TDM were observed. Five cohort studies showed significant associations between achieving target concentrations and clinical success, including suppression of resistance. Studies in this review showed no obvious improvement in clinical outcomes when TDM is implemented. Optimisation and standardisation of carbapenem TDM practice are needed to improve intervention success and enable study synthesis. Further suitably powered studies of standardised TDM are required to assess the impact of TMD on clinical outcomes and AMR

Multilayered lipid membrane stacks for biocatalysis using membrane enzymes

Multilayered or stacked lipid membranes are a common principle in biology and have various functional advantages compared to single lipid membranes, such as their ability to spatially organize processes, compartmentalize molecules and greatly increase surface area and hence membrane protein concentration. Here we report on a supramolecular assembly of a multilayered lipid membrane system in which poly-L-lysine electrostatically links negatively charged lipid membranes. When suitable membrane enzymes are incorporated, either an ubiquinol oxidase (cytochrome bo3 from Escherichia coli) or an oxygen tolerant hydrogenase (the membrane-bound hydrogenase from Ralstonia eutropha), cyclic voltammetry (CV) reveals a linear increase in biocatalytic activity with each additional membrane layer. Electron transfer between the enzymes and the electrode is mediated by the quinone pool that is present in the lipid phase. We deduce by atomic force microscopy, CV and fluorescence microscopy that quinones are able to diffuse between the stacked lipid membrane layers via defect sites where the lipid membranes are interconnected. This assembly is akin to that of interconnected thylakoid membranes or the folded lamella of mitochondria and have significant potential for mimicry in biotechnology applications such as energy production or biosensing