Biology subjects

Walker, D. J. F.

Publications and source records attributed to Walker, D. J. F..

5 recordsLinked to original sources

bioRxiv2021

Direct Observation of Electrically Conductive Pili Emanating from Geobacter sulfurreducens

Geobacter sulfurreducens is a model microbe for elucidating the mechanisms for extracellular electron transfer in several biogeochemical cycles, bioelectrochemical applications, and microbial metal corrosion. Multiple lines of evidence previously suggested that electrically conductive pili (e-pili) are an essential conduit for long-range extracellular electron transport in G. sulfurreducens. However, it has recently been reported that G. sulfurreducens does not express e-pili and that filaments comprised of multi-heme c-type cytochromes are responsible for long-range electron transport. This possibility was directly investigated by examining cells, rather than filament preparations, with atomic force microscopy. Approximately 90 % of the filaments emanating from wild-type cells had a diameter (3 nm) and conductance consistent with previous reports of e-pili harvested from G. sulfurreducens or heterologously expressed in E. coli from the G. sulfurreducens pilin gene. The remaining 10% of filaments had a morphology consistent with filaments comprised of the c-type cytochrome OmcS. A strain expressing a modified pilin gene designed to yield poorly conductive pili expressed 90 % filaments with a 3 nm diameter, but greatly reduced conductance, further indicating that the 3 nm diameter conductive filaments in the wild-type strain were e-pili. A strain in which genes for five of the most abundant outer-surface c-type cytochromes, including OmcS, was deleted yielded only 3 nm diameter filaments with the same conductance as in the wild-type. These results demonstrate that e-pili are the most abundant conductive filaments expressed by G. sulfurreducens, consistent with previous functional studies demonstrating the need for e-pili for long-range extracellular electron transfer. ImportanceElectroactive microbes have significant environmental impacts as well as applications in bioenergy and bioremediation. The composition, function, and even existence of electrically conductive pili (e-pili) has been one of the most contentious areas of investigation in electromicrobiology, in part because e-pili offer a mechanism for long-range electron transport that does not involve the metal co-factors common in much of biological electron transport. This study demonstrates that e-pili are abundant filaments emanating from Geobacter sulfurreducens, which serves as a model for long-range extracellular electron transfer in direct interspecies electron transfer, dissimilatory metal reduction, microbe-electrode exchange, and corrosion caused by direct electron uptake from Fe(0). The methods described in this study provide a simple strategy for evaluating the distribution of conductive filaments throughout the microbial world with an approach that avoids artifactual production and/or enrichment of filaments that may not be physiologically relevant.

microbiology↗

bioRxiv2021

Multi-modal engineering of Bst DNA polymerase for thermostability in ultra-fast LAMP reactions

DNA polymerase from Geobacillus stearothermophilus, Bst DNA polymerase (Bst DNAP), is a versatile enzyme with robust strand-displacing activity that enables loop-mediated isothermal amplification (LAMP). Despite its exclusive usage in LAMP assay, its properties remain open to improvement. Here, we describe logical redesign of Bst DNAP by using multimodal application of several independent and orthogonal rational engineering methods such as domain addition, supercharging, and machine learning predictions of amino acid substitutions. The resulting Br512g3 enzyme is not only thermostable and extremely robust but it also displays improved reverse transcription activity and the ability to carry out ultrafast LAMP at 74 {degrees}C. Our study illustrates a new enzyme engineering strategy as well as contributes a novel engineered strand displacing DNA polymerase of high value to diagnostics and other fields.

bioengineering↗

bioRxiv2020

Cytochrome OmcS is not essential for long-range electron transport in Geobacter sulfurreducens strain KN400

The multi-heme c-type cytochrome OmcS, is one of the central components for extracellular electron transport in Geobacter sulfurreducens strain DL-1, but its role in other microbes, including other strains of G. sulfurreducens is currently a matter of debate. Therefore, we investigated the function of OmcS in G. sulfurreducens strain KN400, which is even more effective in extracellular electron transfer than strain DL-1. We found that deleting omcS from strain KN400 did not negatively impact the rate of Fe(III) oxide reduction and did not affect the strains ability to accept electrons via direct interspecies electron transfer. The OmcS-deficient strain also continued to produce conductive filaments, consistent with the concept that electrically conductive pili are the primary conduit for long-range electron transfer in G. sulfurreducens and closely related species. These findings, coupled with the lack of OmcS homologs in most other microbes capable of extracellular electron transfer, suggest that OmcS is not a common critical component for extracellular electron transfer.

microbiology↗

bioRxiv2018

The Archaellum of Methanospirillum hungatei is Electrically Conductive

Here we report that the archaellum of Methanospirillum hungatei is electrically conductive. Our analysis of the previously published archaellum structure suggests that a core of tightly packed phenylalanines is one likely route for electron conductance. This is the first demonstration that electrically conductive protein filaments (e-PFs) have evolved in Archaea and is the first e-PF for which a structure is known, facilitating mechanistic evaluation of long-range electron transport in e-PFs.

microbiology↗

bioRxiv2017

Electrically Conductive Pili from Pilin Genes of Phylogenetically Diverse Microorganisms

The possibility that bacteria other than Geobacter species might contain genes for electrically conductive pili (e-pili) was investigated by heterologously expressing pilin genes of interest in Geobacter sulfurreducens. Strains of G. sulfurreducens producing high current densities, which are only possible with e-pili, were obtained with pilin genes from Flexistipes sinusarabici, Calditerrivibrio nitroreducens, and Desulfurivibrio alkaliphilus. The conductance of pili from these strains was comparable to native G. sulfurreducens e-pili. The e-pili derived from C. nitroreducens, and D. alkaliphilus pilin genes are the first examples of relatively long (> 100 amino acids) pilin monomers assembling into e-pili. The pilin gene from Desulfofervidus auxilii did not yield e-pili, suggesting that the hypothesis that this sulfate reducer wires itself to ANME-1 microbes with e-pili to promote anaerobic methane oxidation should be reevaluated. A high density of aromatic amino acids and a lack of substantial aromatic-free gaps along the length of long pilins may be important characteristics leading to e-pili. This study demonstrates a simple method to screen pilin genes from difficult-to-culture microorganisms for their potential to yield e-pili; reveals new potential sources for biologically based electronic materials; and suggests that a wide phylogenetic diversity of microorganisms may employ e-pili for extracellular electron exchange.

microbiology↗