Biology subjects

Zacharoff, L. A.

Publications and source records attributed to Zacharoff, L. A..

5 recordsLinked to original sources

bioRxiv2021

Single molecule tracking of bacterial cell surface cytochromes reveals dynamics that impact long-distance electron transport

Using a series of multiheme cytochromes, the metal-reducing bacterium Shewanella oneidensis MR-1 can perform extracellular electron transfer (EET) to respire redox-active surfaces, including minerals and electrodes outside the cell. While the role of multiheme cytochromes in transporting electrons across the cell wall is well established, these cytochromes were also recently found to facilitate long-distance (micrometer-scale) redox conduction along outer membranes and across multiple cells bridging electrodes. Recent studies proposed that long-distance conduction arises from the interplay of electron hopping and cytochrome diffusion, which allows collisions and electron exchange between cytochromes along membranes. However, the diffusive dynamics of the multiheme cytochromes have never been observed or quantified in vivo, making it difficult to assess their hypothesized contribution to the collision-exchange mechanism. Here we use quantum dot labeling, total internal reflection fluorescence microscopy, and single-particle tracking to quantify the lateral diffusive dynamics of the outer membrane-associated decaheme cytochromes MtrC and OmcA, two key components of EET in S. oneidensis. We observe confined diffusion behavior for both quantum dot-labeled MtrC and OmcA along cell surfaces (diffusion coefficients DMtrC = 0.0192 {+/-} 0.0018 m2/s, DOmcA = 0.0125 {+/-} 0.0024 m2/s) and the membrane extensions thought to function as bacterial nanowires. We find that these dynamics can trace a path for electron transport via overlap of cytochrome trajectories, consistent with the long-distance conduction mechanism. The measured dynamics inform kinetic Monte Carlo simulations that combine direct electron hopping and redox molecule diffusion, revealing significant electron transport rates along cells and membrane nanowires. SignificanceMultiheme cytochromes in Shewanella oneidensis MR-1 transport electrons across the cell wall, in a process called extracellular electron transfer. These electron conduits can also enable electron transport along and between cells. While the underlying mechanism is thought to involve a combination of electron hopping and lateral diffusion of cytochromes along membranes, these diffusive dynamics have never been observed in vivo. Here, we observe the mobility of quantum dot-labeled cytochromes on living cell surfaces and membrane nanowires, quantify their diffusion with single-particle tracking techniques, and simulate the contribution of these dynamics to electron transport. This work reveals the impact of redox molecule dynamics on bacterial electron transport, with implications for understanding and harnessing this process in the environment and bioelectronics.

microbiology↗

bioRxiv2021

Abnormal Recovery from Acute Stress in Huntingtons Disease Mice

Introduction: Comparisons of hypothalamic dysfunction between Huntingtons Disease (HD) patients and rodent models of HD have not always yielded similar results. Cortisol levels in HD patients have been contradictory, with reports ranging from hypo- to hypercorticoidism of morning measurements. Initial reports of major elevations in circulating corticosterone levels in the R6/2 mouse model of HD have only been followed up in one other closely related model, the R6/1 mouse, and the results were not perfectly congruent. To determine if abnormal stress hormones were a characteristic of disease, we examined diurnal and stress-induced corticosterone levels in multiple HD mouse models. Methods: We analyzed serum corticosterone levels from R6/2, Q140 and BACHD mice either diurnally (R6/2) or after restraint stress and subsequent recovery (all 3 models). Glucocorticoid secretion was also examined (R6/2) following dexamethasone suppression and ACTH stimulation. Blood samples were collected either by submandibular puncture or as trunk blood at sacrifice. Plasma corticosterone levels were assayed by RIA. Regional brain glucocorticoid receptor (GR) levels were visualized using immunofluorescence. Results: In the R6/2 mouse, basal levels of corticosterone, exhibited normal, sexually dimorphic diurnal rhythms but eventually became elevated towards end of life, after 13 weeks. The ability of the adrenal gland to produce corticosterone was not altered and the adrenal response to ACTH stimulation was only elevated in male R6/2 mice compared to littermates. Down regulation of GR receptors was only observed in the PVN at 16 weeks. R6/2 mice at both 6 and 12 weeks responded equivalently to littermates in the initial corticosterone response to restraint stress but serum corticosterone recovered from stress significantly more slowly. No differences were found in the initial stress response or in the recovery from stress for Q140 or BACHD mice, examined at 12 and 9 months, respectively. Conclusions: We did not find evidence of major disruption in glucocorticoid signaling in HD mouse models. A significantly slowed recovery from stress observed in the R6/2 mouse suggests such disruptions are context dependent. These results suggest that disruption of the HPA axis was subtle and occurred only at advanced stages of disease represented by comparisons among these HD mouse models.

neuroscience↗

bioRxiv2021

In situ imaging of bacterial membrane projections and associated protein complexes using electron cryo-tomography

The ability to produce membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of [~] 90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified MEs and MVs in 13 species and classified several major ultrastructures: 1) tubes with a uniform diameter (with or without an internal scaffold), 2) tubes with irregular diameter, 3) tubes with a vesicular dilation at their tip, 4) pearling tubes, 5) connected chains of vesicles (with or without neck-like connectors), 6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.

molecular biology↗

bioRxiv2020

A Prokaryotic Membrane Sculpting BAR Domain Protein

Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here we report the first prokaryotic BAR domain protein, BdpA, from Shewanella oneidensis MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and scaffolding OMEs into a consistent diameter and curvature. Cryogenic transmission electron microscopy reveals a strain lacking BdpA produces lobed, disordered OMEs rather than membrane tubes produced by the wild type strain. Overexpression of BdpA promotes OME formation during conditions where they are less common. Heterologous expression results in OME production in Marinobacter atlanticus and Escherichia coli. Based on the ability of BdpA to alter membrane curvature in vivo, we propose that BdpA and its homologs comprise a newly identified class of prokaryotic BAR (P-BAR) domains.

microbiology↗

bioRxiv2017

Geobacter sulfurreducens extracellular multiheme cytochrome PgcA facilitates respiration to Fe(III) oxides but not electrodes

Extracellular cytochromes are hypothesized to facilitate the final steps of electron transfer between the outer membrane of the metal-reducing bacterium Geobacter sulfurreducens and solid-phase electron acceptors such as metal oxides and electrode surfaces during the course of respiration. The triheme c-type cytochrome PgcA exists in the extracellular space of G. sulfurreducens, and is one of many multiheme c-type cytochromes known to be loosely bound to the bacterial outer surface. Deletion of pgcA using a markerless method resulted in mutants unable to transfer electrons to Fe(III) and Mn(IV) oxides; yet the same mutants maintained the ability to respire electrode surfaces and soluble Fe(III) citrate. When expressed and purified from Shewanella oneidensis, PgcA demonstrated a primarily alpha helical structure, three bound hemes, and was processed into a shorter 41 kDa form lacking the lipodomain. Purified PgcA bound Fe(III) oxides, but not magnetite, and when PgcA was added to cell suspensions of G. sulfurreducens, PgcA accelerated Fe(III) reduction similar to addition of FMN. Addition of soluble PgcA to {triangleup}pgcA mutants also restored Fe(III) reduction. This report highlights a distinction between proteins involved in extracellular electron transfer to metal oxides and poised electrodes, and suggests a specific role for PgcA in facilitating electron transfer at mineral surfaces.

microbiology↗