Cation-driven envelope dynamics modulate outer membrane vesiculation and extracellular electron transfer in Geobacter

Cation-driven envelope dynamics modulate outer membrane vesiculation and extracellular electron transfer in Geobacter | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cation-driven envelope dynamics modulate outer membrane vesiculation and extracellular electron transfer in Geobacter Gemma Reguera, Morgen Clark, Kazem Kashefi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8864095/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Geobacter bacteria use conductive pili and redox-active outer membrane vesicles to mediate metal transformations critical to the effectiveness of bioremediation and energy technologies. Mechanistic knowledge into these processes primarily comes from studies with Geobacter sulfurreducens grown in media closely formulated to mirror the mineral chemistry of contaminated sites. Although subtle differences in the media’s cationic strength did not measurably change permeability, they reprogrammed outer membrane-peptidoglycan crosslinks modulating vesiculation and envelope functions impacting growth efficiency and mineralization. Cations that strongly bind and neutralize peptidoglycan carboxylates to prevent cell wall distortions, such as sodium and uranyl ions, ultimately determined the extent of envelope remodeling and cell bias toward pili-driven mineralization or membrane adsorption and release in vesicles. These findings identify cation chemistry as a key regulator of outer membrane vesiculation and the reprogramming of envelope functions ultimately determining the reproducibility of laboratory studies and effectiveness of bioremediation and energy-harvesting applications. Biological sciences/Microbiology/Bacteria/Bacterial physiology Earth and environmental sciences/Biogeochemistry/Element cycles Outer membrane vesicles peptidoglycan crosslinks Gram-negative envelope microbial nanowires Type IV pili Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The biomineralization of iron and other metals by electrically active (electric) microorganisms represents one of the oldest and most influential geobiological processes on Earth 1 – 3 . These microbes, and the conductive minerals they generate, establish natural electric grids that metabolically couple microbial communities and shape the geochemistry of entire ecosystems 4 – 7 . Among them, members of the Geobacterales provide foundational models for understanding how microbial electrical networks drive metal 1 and, indirectly, carbon 8 sequestration into stable mineral sinks and how their metabolisms can be harnessed to mine and reclaim metals critical to energy storage technologies 9 . These electrical activities are primarily driven by a trans-envelope respiratory chain of c- cytochromes 10 as well as retractable, conductive pili assembled on one side of the cell to extend the redox-active surface beyond outer membrane c -cytochromes. 11 , 12 The conductive pili shared many of the conserved properties of bacterial Type IVa pili, yet are assembled with a uniquely short, 11 aromatically dense, 13 and intrinsically conductive 14 pilin peptide. The ubiquity of similar, aromatically dense pilin sequences in metal-rich environments 15 suggests that pili-mediated electron flow is a widespread mechanism for metal cycling across ecosystems. Pilus nanowires also drive the reductive mineralization of soluble toxic metals and radionuclides such as the uranyl oxycation (UO 2 2+ ). 16 Surface motifs of anionic ligands promote the binding of cationic metals to the pilus fibers to facilitate their reductive mineralization, 16 in a process that also conserves energy for growth. 17 Pilus nanowires also permeate the exopolysaccharide (EPS) matrix of biofilms, establishing vertical redox gradients with EPS-anchored c -cytochromes for optimal electron flow 18 and enhancing uranium mineralization. 19 Field amendments with electron donors such as acetate stimulate these activities and promote the in situ immobilization of uranium and other toxic metals in contaminated environments. 20 – 24 Guiding these and other applications are laboratory studies with representative strains, primarily Geobacter sulfurreducens , grown in bicarbonate-based mineral media formulated with bicarbonate and mineral salts that replicate the geochemistry of contaminated sites. 25 , 26 These studies identified a complementary pathway for the detoxification of uranyl cations via their adsorption to the rough (no O-antigen) lipopolysaccharide (LPS) that densely coats Geobacter cells and the disposal of LPS-bound metal in outer membrane vesicles (OMVs) 27 ( Fig. S1 ). Vesiculation also provides a mechanism for the detoxification of any uranium which may have diffused and mineralized in the periplasm 16 due to the high-affinity and low-potential of periplasmic c -cytochromes 28 ( Fig. S1 ). The presence of c -cytochromes in the OMVs also suggests roles in extracellular electron transfer to minerals and other cells in biofilms. 29 , 30 Furthermore, OMVs readily attach to solid surfaces and lower interfacial resistance. 29 Thus, the vesicles might complement or amplify mineralization reactions driven by the pili. Cultivation conditions strongly bias whether cells assemble pili or vesiculate, thus undermining the reproducibility and predictive power of laboratory studies. For examples, pilus nanowires are required for iron or manganese oxides respiratory growth but not for the respiration of soluble electron acceptors such as fumarate. 11 Yet, non-piliated cells can grow optimally in fumarate cultures under uranium stress by hypervesiculating. 27 Furthermore, vesiculation is attenuated in the fumarate-grown cultures at temperatures (25 o C) that slow down growth as in cultures with metal oxides and induce pili assembly. 11 , 16 These reciprocal responses suggest that pili-wired and OMV-based detoxification pathways are co-regulated. Evidence further implicates outer membrane-peptidoglycan crosslink rearrangements in this coordination. Genetic disruption of one of several outer membrane OmpA-like anchors in G. sulfurreducens increases both vesiculation and current harvesting from electrode-associated biofilms. 30 Moreover, the hypervesiculating cells release OMVs enriched in OmcZ, 30 an outer membrane c -cytochrome that accumulates closer to the electrode surface to lower interfacial resistance. 31 Yet as the biofilm cells grow away from the electrode, the wiring capacity of the conductive pili becomes increasingly more important 18 , enhancing current harvesting 18 and uranium detoxification. 19 These observations point to a coordinated regulation of pili- and OMV-based electron transfer pathways via yet unresolved envelope mechanisms. Motivated by this gap in knowledge, we investigated how G. sulfurreducens modulates vesiculation when grown in two mineral media (NB and DB) indistinctively used for Geobacter cultivation. NB, a nutrient broth originally formulated for genetic studies, 32 remains a popular medium for metal reduction and OMV research. 27 , 29 , 30 DB is an NB-derived mineral formulation originally used for electrochemical studies of electrode-immobilized cells 33 used, once supplemented with the NB vitamin mix, for electrochemical 34 and metal detoxification 35 growth studies. Both media use bicarbonate buffering to recreate the complexation of cationic metals in the contaminated subsurface. 25 , 26 Yet, as we show, previously overlooked differences in the media cationic strength, particularly from sodium (Na + ), influence outer membrane-peptidoglycan crosslinks and reciprocally regulate piliation and vesiculation. These findings refine current models of OMV control in Gram-negative bacteria and reveal physicochemical drivers of envelope remodeling that ultimately determine the reproducibility of laboratory phenotypes and metal transformations driven by Geobacter bacteria. We further discuss the implications of these findings for translating laboratory observations into field-scale and energy-harvesting applications with Geobacter . Results Control of outer membrane vesiculation by Na + ionic strength. In order to standardize culture conditions for the study of vesiculation in G. sulfurreducens , we quantified OMVs in mid-exponential acetate-fumarate cultures using two popular mineral media (NBAF 32 and DBAF, 34 respectively, but abbreviated as NB and DB herein). Incubation was at 35 o C to support optimal planktonic growth and prevent piliation. 11 , 16 OMV detection leveraged the high adsorption capacity of highly-oriented pyrolytic graphite (HOPG) for extracellular vesicles and the imaging power of an atomic force microscope (AFM) 36 to track vesiculation levels in culture samples, as previously described. 27 The method also avoided the artefacts introduced with protocols requiring OMV extraction, purification, and single-particle tracking analysis, 37 previously applied to characterize G. sulfurreducens OMVs. 29 , 30 Although the NB and DB formulations contained comparable levels of sodium bicarbonate (21.4 and 23.8 mM NaHCO 3 , respectively) and were identically buffered (pH of 6.8) and amended with vitamins, their salt content differed subtly ( Table S1 ). Most notably, the DB medium lacked two weakly complexed Na + salts (6 mM NaCl and 3.5 mM Na 2 CO 3 ) from the NB formulation, decreasing the molar concentration of the alkali cation in DB from 89.5 to 79.3 mM. This difference markedly influenced the vesiculation response, stimulating it in the DB cultures (Fig. 1 ) despite measurably impacts on outer membrane permeability ( Fig. S2A ). However, addition of one or two of the NB salts to the DB cultures chemically rescued the hypervesiculation phenotype (Fig. 1 A). Overall, average OMV yields per cell linearly scaled with the molar Na + concentration in the cultures (R 2 = 0.999). Further supporting the direct role of Na + in vesiculation control, OMV formation in the NB cultures was further attenuated by adding yeast extract ( Fig. S3 ), a concentrate of the soluble fraction of osmotically autolyzed yeast that carries high molar concentrations of Na + salts 38 and is commonly added to G. sulfurreducens cultures. 39 By contrast, NB supplementation with cysteine, a reducing agent commonly added to ensure anoxic conditions, 32,40 had no effect on OMV release ( Fig. S3 ). These results suggested that Na + ionic strength is a key modulator of outer membrane vesiculation in G. sulfurreducens . Further supporting this, increasing the concentration of sodium acetate as an electron donor did not significantly impact vesiculation until the weakly complexed NB salts were added ( Fig. S2 ), consistent with the stronger binding of Na + to acetate (CH 3 COO⁻) than to chloride (Cl – ) or carbonate (CO 3 2– ) anions predicted from the Hofmeister series. 41 By contrast, the type of alkaline ion mattered and higher concentrations of K + than Na + chloride and carbonate salts were needed to attenuate the hypervesiculation response of DB-grown cells to NB levels ( Fig. S2 ). This is consistent with a mechanism for vesiculation control mediated by electrostatic interactions that sterically favored the smaller Na + cation. Na + ionic strength remodels outer membrane-peptidoglycan crosslinks and respiratory chains. What type of electrostatic interactions with Na + could modulate OMV formation? This alkali monovalent cation, more so than K + , diffuses freely through the outer membrane of Gram-negative cells 42 and strongly binds the peptidoglycan’s carboxylate groups, 43 minimizing repulsion forces that would otherwise cause cell wall expansions. 44 Consistent with this, cryo-EM imaging ( Fig. S4 ) revealed enlarged periplasms (Fig. 1 C) and, accordingly, larger cell surface area (measured by AFM, Fig. 1 D) in DB-grown cells compared to those from NB or salt-amended DB cultures (Fig. 1 D). Cell size also scaled linearly with OMV formation across all culture conditions ( Fig. S5 ), strengthening the link between Na + -driven cell wall dynamics and vesiculation control. We gained further insights into envelope adaptations to Na + by comparing the proteomes ( Data S1 ) and transcriptomes ( Data S2 ) of DB-grown cells to NB controls. Though relatively modest (~ 10 mM), differences in Na + molar concentrations in the two media drove broad changes in envelope-associated proteins ( Fig. S6-7 ), including lipoproteins tethering the outer membrane to the underlying peptidoglycan (Fig. 2 ). The most significant change was the upregulation in the DB-grown cells of Lpp, the abundant Braun lipoprotein that covalently crosslinks the outer membrane to the peptidoglycan to preserve its integrity 45 and mechanically stabilize the cell envelope. 46 The Lpp of G. sulfurreducens is also longer than canonical Lpp proteins and well suited to accommodate periplasmic enlargements, 47 such as those observed in the DB-grown cells (Fig. 1 ). Pba, one of several OmpA-like p eptidoglycan- b inding protein a nchors implicated in vesiculation control in G. sulfurreducens 30 and other Gram-negative bacteria, 48 was significantly downregulated in the transcriptomic dataset and, to a lesser extent, in the proteome (significance after imputation of a missing value was p = 0.12) (Fig. 2 A). Proteins of the Type IV pilus machinery (PilM) and the structural subunit of the conductive pili (PilA pilin) 11 were also upregulated in the DB proteomes (Fig. 2 A). The trans-envelope pilus complex not only enhances the respiratory capacity of the cells, 11 it also reinforces outer membrane-peptidoglycan crosslinks 49 , 50 . DB-grown cells also upregulated other proteins involved in extracellular electron transfer ( Table S2 ), most notably the XapD ABC transporter and XapK glycosyltransferase required for the synthesis of the redox-active Xap EPS 51 and the small PgcA c -cytochrome that facilitates interfacial electron transfer with minerals 52 (Fig. 2 A). Transcriptional profiling ( Fig. S7 ) revealed additional reprogramming of membrane-bound proteins not captured within the significant thresholds set for the proteomic data (Fig. 2 B and Table S3 ). These proteins included outer membrane c -cytochromes involved in iron oxide reduction such as OmcS 53 and those (OmcG and OmcH) involved in their regulation. 10 Many of these c -cytochromes are enriched in OMVs 29 , 30 and could have been underrepresented in the cells harvested for quantitative proteomics (Fig. 2 A) and heme-staining detection ( Fig. S7 ). Heme-staining is further compounded by the similar electrophoretic mobility of many envelope c -cytochromes, 54,55 which can mask expression changes. Similarly, transcriptomics but not proteomics analysis revealed the upregulation in DB-grown cells of the ATP-binding subunit of an ABC exporter (YbhF) with predicted roles in Na + :H + antiport 56 and the e xtra c ytoplasmic f unction (ECF) sigma factor RpoE ( Fig. S7 ), an alternative sigma factor that modulates the expression of pilus nanowires and c -cytochromes in G. sulfurreducens. 57 The functional significance of these transcriptional shifts in cation-driven envelope remodeling remains to be determined. Notably, RpoE regulates OMV production and other envelope responses in numerous Gram-negative bacteria, 58 yet is role in G. sulfurreducens is largely unexplored. This highlights the need for further studies to determine how this bacterium reprograms envelope responses to cation-driven structural adaptations. Envelope adaptations to the uranyl oxycation reveal dynamic trade-offs between vesiculation and piliation. Like Na + , the uranyl oxycation also strongly binds and effectively neutralizes the peptidoglycan carboxylates. 43 Consistent with this, culture supplementation with uranyl acetate (DB + U and NB + U ) abolished differences in the periplasmic width and surface area of the DB- versus NB-grown cells (Fig. 3 A). The average periplasmic width of uranium-treated cells in both media was among the largest measured (~ 30–32 nm), as expected from the complexation of the peptidoglycan carboxylates with the much larger uranyl oxycation. However, the average size of uranium-treated cells was smaller than in untreated cultures ( Fig. S8A ), suggesting that peptidoglycan complexation with the rigid, linear uranyl oxycation (O = U=O 2+ ) imposed constrains to overall cell expansions. Uranium supplementation also triggered piliation in both cultures, albeit more strongly in the DB + U than in the NB + U cultures (14.4% of the cells had extensive mineralization compared to 8.5% in NB + U ) (Fig. 3 B-C). AFM scans showed the characteristic monolateral mineralization of uranium by piliated cells 16 in the cultures and identified, at closer magnification, uranium nanoparticles permeated by pilus fibers with the characteristic AFM height (2.5 nm) of uranium-reducing pili 13 , 59 ( Fig. S9 ). Furthermore, uranium-treated cells agglutinated ( Fig. S8B ), an aggregative phenotype associated with pili production. 60 Thus, uranyl cations not only counteracted the structural effects of Na⁺ ionic strength but also shifted the cellular response toward enhanced piliation and mineralization. AFM scans of uranium-treated cultures revealed OMVs with a similar AFM diameter (~ 10 nm) as in the untreated cultures ( Fig. S9 ). However, vesiculation yields were reversed in uranium-treated cultures, decreasing in DB + U and increasing in NB + U (Fig. 3 B). Furthermore, OMV release per cell inversely correlated with the levels of piliated cells in the uranium-treated cultures (Fig. 3 C). These findings suggest dynamic trade-offs between pili and OMV pathways during uranium detoxification, where ionic conditions favoring piliation attenuate vesiculation and vice versa. Yet both mechanisms are needed to protect the cell from uranyl toxicity. This is because pilus nanowires confine mineralization to a single side of the cell, 11,16 leaving the opposite side reliant on uranyl binding to its rough LPS layer and detoxification via OMV release. 27 Differential impact of Na + and uranyl cations on growth and metal mineralization. Rigorous control of media preparation and culture passage ensured phenotypic reproducibility and unmasked subtle, yet significant, differences in growth efficiency as a function of cation chemistry (Na + and uranyl) (Fig. 4 A). The DB-grown cells reproducibly underwent slightly longer (> 1 hour) lag phases yet, once acclimated, they doubled faster than in the NB medium. Moreover, growth yields measured by optical density were similar in both cultures but masked the lower viability of the DB cells upon entry into stationary phase. Mid-exponential DB cells also experienced more viability losses upon growth arrest in a bicarbonate-buffered solution (Fig. 4 B). Additionally, the enzymatic removal of uranyl acetate per viable cell in the resting state was higher when using DB-grown cells compared to NB (Fig. 4 B). The upregulation in the DB cells of envelope proteins for extracellular electron transfer such as pili and c- cytochromes (Fig. 2 ) could have accounted for these differences, as previously shown. 16 Cell wall and periplasmic expansions in the DB-grown cells (Fig. 1 ) may have also made more peptidoglycan carboxylates available for binding the uranyl cations and prime them for more efficient uranium removal once in the resting state. Ultimately, these results caution about the importance of cultivation conditions in the metal-reducing phenotypes reported in resting cell assays, whose reproducibility depends on respiratory capacity 61 and cellular integrity. 62 We also compared the growth efficiency of G. sulfurreducens in NB or DB cultures supplemented with 500 µM uranyl acetate (NB + U and DB + U cultures) (Fig. 4 A). The NB + U and DB + U cultures acclimated and grew at comparable rates in the presence of uranium, albeit less efficiently than in untreated controls ( Fig. S10 ). However, growth yields (both by optical density and viable cell counts) were significantly lower in the DB + U medium compared to NB + U (Fig. 4 A). The heightened susceptibility of growth-arrested DB cells to uranium likely reflects the compounded effects of uranium-induced envelope stress on cells already compromised by low Na⁺ ionic strength, which makes their envelope more vulnerable to uranium toxicity once growth is arrested and active mechanisms for detoxification and repair are no longer active. Discussion Findings from this work demonstrate that cation chemistry is a key and previously underappreciated regulator of outer‑membrane vesiculation and envelope remodeling in Gram-negative bacteria. Subtle, yet physiologically relevant, differences in the medium’s Na⁺ ionic strength remodeled outer membrane-peptidoglycan crosslinks, adjusted the periplasmic volume and cell size, and modulated vesiculation in G. sulfurreducens (Fig. 1 ). These adaptive responses to Na + ionic strength reflect the free diffusion of the alkali cation through the outer membrane, 42 its strong affinity for peptidoglycan carboxylates, 43 and its ability to neutralize carboxylate charges to prevent repulsion forces that would otherwise cause cell wall expansions. 44 Insufficient free Na + in the DB medium led to cell wall expansions that widened the periplasm and destabilized non-covalent tethering via Pba, inducing curvature stress and vesiculation, even though the outer membrane-peptidoglycan lattice was covalently reinforced with Lpp anchors (Figs. 1 and 2 ). However, culture supplementation with weakly complexed Na + salts alleviated these constraints and chemically rescued the hypervesiculating phenotype of DB-grown cells (Fig. 1 ). K + similarly modulated OMV formation but less effectively than Na + due its larger ionic radius and lower ionization potential, 63 which makes local ion pairing with peptidoglycan carboxylate ligands more inefficient. 64 These results highlight how steric and electrostatic factors unique to each alkali metal ion contribute to envelope remodeling. Conversely, the inability of additional sodium acetate to suppress vesiculation in DB cultures ( Fig. S2 ) underscores how anion pairing can strongly limit free Na⁺ and cation-driven envelope adaptations. Control of vesiculation via cation-driven remodeling of envelope crosslinks complements genetic studies linking outer membrane-peptidoglycan tethers to OMV formation, particularly those mediated by non-covalent OmpA-like anchors such as Pba 30 , 48 and the Braun lipoprotein Lpp. 65 Importantly, because vesiculation control in G. sulfurreducens only required subtle changes in cation chemistry, we circumvented the broad pleiotropic defects reported for anchor mutants 48 and instead examined envelope adaptations under physiological relevant conditions. As a mechanically tethered system, 66 the outer membrane-cell wall lattice synchronously responds to cation deficiencies via peptidoglycan expansions that widen the periplasm. 67 , 68 G. sulfurreducens adapted to the periplasmic enlargements in DB cultures by making more Lpp (Fig. 2 ), a trimeric anchor that covalently reinforces outer membrane-peptidoglycan crosslinks to stabilize the envelope. 69 , 70 By transitioning from tilted to stretched triple coiled-coil configurations, the flexible Lpp trimer stabilizes the envelope and preserves the integrity of the outer membrane during periplasmic enlargements. 47 This capacity is further enhanced in Geobacter due to its unusually long Lpp. 47 Studies also show that the tilted-to-stretched transition of the Lpp trimer during periplasmic enlargements quickly dislodges non-covalent Pba anchors, 47 leaving untethered areas more prone to vesiculation and producing undulating membrane features, the same phenotypes we observed in the DB-grown cells (Fig. 1 ). The reciprocal regulation of Pba and Lpp anchors likely arises as a compensatory response to the structural remodeling of cross-links triggered by low ionic strength. The possibility of fine-tuning these crosslinks and regulatory feedbacks via adjustments in the culture’s Na + ionic strength makes Geobacter a powerful system to study the plasticity of the Gram-negative envelope to spatial constraints imposed by chemical cues. Like Na + , the uranyl oxycation (UO₂²⁺) exhibits a strong affinity for peptidoglycan carboxylate groups, 43 effectively neutralizing their negative charges and counteracting the differential effects of Na + ionic strength on periplasmic and cell surface area expansions (Fig. 3 ). Nevertheless, uranium imposes additional physicochemical constraints on the envelope due its larger size, rigid linear structure (O = U=O 2+ ), strong bidentate bonds, and ability to form polynuclear U(VI) complexes with carboxylates that yield a rigid and expanded uranium-peptidoglycan scaffold. 43 Consequently, uranium-treated cells had, on average, larger periplasms than untreated controls (Fig. 3 ). Yet their cell size was constrained by the rigidity of the cell wall ( Fig. S8 ). These physicochemical properties also promoted robust assembly of the Type IV pilus machinery for the reductive mineralization of uranium (Fig. 3 ). The slightly enlarged periplasms of the uranium-treated cells likely alleviated spatial barriers to the orderly assembly and alignment of pre-pilus intermediates across the Geobacter multi-layered envelope, 71 whereas the more rigid peptidoglycan scaffold provided mechanical support to the peptidoglycan anchors of the pilus complex. 49 , 50 As a result, G. sulfurreducens pili can more effectively counteract the destabilizing forces imposed by the antagonistic cycles of pilus protrusion and retraction that are key to effective uranium mineralization and respiratory gains. 12 However, DB + U cultivation, more so than NB + U , markedly increased piliation, consistent with compounding effects of peptidoglycan-binding cations on envelope remodeling. However, cation‑driven changes in the envelope imposed a dynamic tradeoff between pili‑mediated mineralization and OMV‑mediated export, reciprocally regulating the two pathways (Fig. 3 ). This balance likely reflects the compounding effects of steric crowding and stronger tethering of the outer membrane in piliated cells, which spatially and structurally limits OMV biogenesis. Such constrains reciprocally coordinate the selection of pathways during metal detoxification, preventing envelope stress yet having opposing impacts on respiratory gains and metal transformations (pili-mediated mineralization versus reversible adsorption to vesicles). Taken together, our results demonstrate that cation chemistry is an unappreciated experimental variable that must be rigorously controlled to ensure reproducibility in studies of extracellular electron transfer and metal transformations. Many discrepancies in the Geobacter literature, and likely in other Gram‑negative systems, might be traced to unstandardized cations (e.g., Na⁺, K⁺) and associated counterions in media formulations and, potentially, other cultivation variables modulating piliation such as temperature. 1 Genetic analyses in Geobacter further confound experimental results across labs due to the pleiotropic nature of most disruptions of envelope proteins, particularly pili and outer‑membrane c -cytochromes. 16 , 18 In contrast, chemical modulation of envelope mechanics provides a powerful and physiologically relevant tool to dissect these pathways without exacerbating compensatory responses. The compounding effects of cation-driven envelope adaptations on bioremediation outcomes and the performance of energy-harvesting technologies driven by Geobacter are yet to be addressed. Moreover, the diverse and poorly understood repertoire of OmpA-like anchors in G. sulfurreducens 30 represents an opportunity to dissect how chemical cues selectively bias envelope crosslinks toward piliation or vesiculation. Given the roles of OMVs in transporting c -type cytochromes and modulating interfacial electron transfer reactions in Geobacter 29 , 30 and their ability to form conductive nanotubes in other bacteria 72 accounting for local ionic conditions will prove essential for producing reproducible datasets that can meaningfully guide bioremediation and energy systems driven by Geobacter . Materials and Methods Bacterial strains and culture conditions. G. sulfurreducens PCA 73 and ∆ omcS 53 and ∆ omcB mutants 74 from our culture collection were routinely grown at 35 o C in NBAFYE 32 , a nutrient broth (NB) medium with acetate (15 mM), fumarate (40 mM), cysteine (1 mM) and yeast extract (0.1%, w/v). After three transfers (starting OD 600 of 0.01–0.03) in mid-exponential phase (0.4–0.6 OD 600 ), the cells were inoculated in test media: NBAF (no cysteine or yeast extract) or a modified media formulation DBAF. 34 When indicated, cultures were amended with various salts (NaCl, Na 2 CO 3 , KCl, and/or potassium phosphate salts) or with 500 µM uranyl acetate. Growth parameters (length of lag phase, generation time, and maximum growth yields) were calculated using the growthcurver package (v0.3.1) in R. Pili-mediated agglutination in the cultures was measured as the OD 600 difference before and after gentle tube inversion. 60 Microscopy. Mid-exponential cultures were routinely imaged in tapping mode at 0.3 Hz with an Asylum Research Cypher S Atomic Force Microscope (AFM) system equipped with an AC240TS tip to count OMVs 27 and uranium-mineralizing cells expressing pili (AFM amplitude images analyzed with the multi-point tool in ImageJ). OMV and pilus filament diameters were measured as AFM height with the Igor Pro (6.38B01) and Asylum AFM software (v16.31.232). The area of the cells in AFM Zsensor images was measured with the “analyze particles” tool of ImageJ, as described in the Supplementary materials. Cryo-EM imaging used a Thermo Scientific™ Talos™ Arctica™ instrument outfitted with a Falcon 3 Direct Detection Device and a Ceta CCD camera and operated at 200 kV. Mid-exponential cell suspensions concentrated via centrifugation (5000x g for 10 min) were deposited (3–5 µl) on 300 copper mesh grids coated with a Quantifoil™ R 3.5/1.0 carbon film (45 sec of negative glow discharge with a Ted Pella Pelco easiGlow), blotted (2.5 and 10 sec blot and wait times, respectively; blot force, -2; humidity, 100%; and temperature, 4°C), and vitrified in liquid ethane in a Vitrobot Mark IV system (ThermoFisher Scientific). Image acquisition used the EPU (Thermo Scientific) software in linear mode at a 22,000x magnification (pixel size 6.6 Å) with a defocus range of − 10 µm and a dose rate of 3.3 e/Å 2 /s for ~ 2 sec. Periplasm widths were measured in ImageJ (“Analyze>Measure” function) by fitting a rotated rectangle (~ 5 nm wide) on the envelope every 15–20 nm, excluding the mid-center and poles (each ~ 1/5th of the cell length). Fluorescence assays of outer membrane permeability and cell viability. Mid-exponential cells were harvested by centrifugation (8000x g , 7 min), washed twice (5 mM HEPES-5 mM glucose buffer pH 7.2), and resuspended in ½ vol of the same buffer before dispensing 100 µl of sample and buffer controls (blanks) in triplicate wells of a white-walled, clear-bottom 96-well plate (Alkali Scientific). A stock of 500 M N-Phenyl-1-naphthylamine (NPN) (Sigma-Aldrich) in acetone was diluted to 20 µM in assay buffer before adding 100 µl to each well, pipetting to mix, and incubating in the dark (room temperature, 10 min) before measuring (350 nm excitation, 420 nm emission) fluorescence from the hydrophobic fluorescent NPN probe after intercalation into the outer membrane phospholipids. 75 , 76 Cell viability was assayed with the Live/Dead® BacLight™ Bacterial Viability Kit (ThermoFisher Scientific) in cells harvested by centrifugation (5,000x g for 10 min) from 0.5-1 ml early-stationary phase cultures or resting cell suspensions, washed twice in 0.85% NaCl, and dispensed (100 µl) in triplicate wells of a 96-well plate (Wuxi NEST), avoiding peripheral wells (rows A and H, columns 1 and 12) for improved reproducibility. A 2X staining solution (4 µl of 5 mM Syto9 and 6 µl of 20 mM propidium iodide per 2 ml nuclease-free water) was added to each sample, pipetting to mix, before dark incubation (room temperature, 15 min) and fluorescence measurements in the green (485/530nm excitation/emission) and red (485/630nm excitation/emission) channels. The ratio of green over red fluorescence was used as a proxy of cell viability. Resting cell assays with uranium. Resting cell suspensions (OD 600 0.1) used mid-exponential cells osmotically adjusted in sequential steps to maximize viability, as previously described 16 , 77 except that the suspensions were prepared in 20 ml of buffer with 500 µM of uranyl acetate. Samples collected before or after 6-h incubation (35 o C) were filter-sterilized (0.22 µm), acidified in 0.8 N nitric acid, and diluted 200-fold in 3% nitric acid before measuring the uranium concentration in an Agilent 8900 triple quadrupole tandem inductively coupled proton mass spectrometer (QQQ-ICP-MS). Proteomics and RNA-Seq analysis. Proteins and RNA transcripts extracted from triplicate 10-ml, mid-exponential NBAF and DBAF cultures were sequenced at Michigan State University Research Technology Support Facility (RTSF) and the SeqCenter (Pittsburg, PA), respectively, following standardized protocols. Protein extraction was from cells harvested by centrifugation (3,220x g , 20 min) and washed three times in 10 mM Tris-HCl buffer (pH 7.7) before resuspending in 500 µl of buffer and centrifuging again in 2-ml cryotubes (8,000x g , 10 min) to obtain a pellet for storage at -80 o C. Cell lysis was initiated in 2x SDS-PAGE sample buffer (125 mM Tris, pH 6.8, 4% SDS, 20% glycerol, 50 mM DTT; 95 o C for 5 min) and, after cooling to room temperature, continued in a water bath sonicator (3 cycles of 1 min sonication and 1 min rest). Protein (100 µg, measured with Pierce BCA assay) was digested with trypsin/LysC (Promega) using S-traps (ProtiFi) according to manufacturer’s instructions before resuspending 20 µl of the digest in 2% acetonitrile/0.1% trifluoroacetic acid and calculating the final peptide concentration with a Pierce Quantitative Colorimetric Peptide Assay Kit. Samples were then run through Data-Independent Acquisition (DIA) LC/MS/MS to obtain the peptide spectra and analyzed as described in the Supplementary Methods and elsewhere. 78 , 79 Missing (‘NA’) values were separately imputed in NB and DB replicates with the R studio missForest package (v1.5). 80 Differentially expressed proteins (DEPs) ( Data S1 ) were determined in Microsoft Excel, as described in the Supplementary Methods. RNA extraction and purification was as previously described. 35 Illumina library preparation and sequencing (paired end 150bp reads on a NovaSeq X Plus instrument) used protocols standardized at SeqCenter. Demultiplexing, quality control, and adapter trimming used Illumina’s bcl-convert (v4.2.4) software, whereas read mapping used HISAT2 (v2.2.0) with default parameters and ’--very-sensitive’. 81 Read quantification used Subread’s featureCounts (v2.0.1) functionality with default parameters and ‘-Q20’. 82 Read counts loaded into R (v4.0.2) were normalized using edgeR’s (v1.14.5) Trimmed Mean of M values (TMM) algorithm with default parameters 83 before conversion to counts per million (CPM). Differential expression analysis used edgeR’s glmQLFTest. The data was then filtered for a |log 2 FC| > 0.58 and p -value < 0.05 to determine differentially expressed genes (DEGs) ( Data S2 ). Adjusted p -values were not used given the low number of DEGs. Functional assignments to DEPs and DEGs used the KEGG and UniProt 84 databases. Analysis of transmembrane regions and subcellular localization was via DeepTMHHM (v1.0) and PSORTb (v3.0.3), respectively. Data visualization (heatmaps, dispersion plots, and volcano plots) used the pheatmap (v1.0.12) and ggplot2 (v3.5.1) packages in R studio. Statistical Analysis. The statistical significance ( p < 0.05) of phenotypic differences in the various cultures was assessed with either a Student’s or Welch’s t -test (chosen based on the results of a F-test for variance) in Excel, as described in the corresponding figure legends. Where indicated, we used the Grubbs’ test for outliers from GraphPad’s online outlier calculator to remove up to one outlier per condition. Data and materials availability. All data are available in the main text or the supplementary materials. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD069850. The RNA-Seq raw data are available at the NCBI Gene Expression Omnibus (GEO) repository under accession number GSE285971. Declarations Data and materials availability All data are available in the main text or the supplementary materials. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD069850. The RNA-Seq raw data are available at the NCBI Gene Expression Omnibus (GEO) repository under accession number GSE285971. Acknowledgments This work was funded by U.S. Army Research Office grant 80459BB to GR and KK) and by National Science Foundation Emerging Frontiers in Research & Innovation grant 2318057 to GR. We are grateful to Dr. Thomas J. Silhavy at Princeton University for discussions of preliminary data inspiring this research and to various colleagues at Michigan State University for technical support in various areas (Dr. Marcela Tabares, RNA-Seq; Nicholas Tefft and Dr. Michaela TerAvest, heme-stained gels; Dr. Sundharraman Subramanian, cryo-EM; and Douglas Whitten, proteomics). We also acknowledge Michigan State University Quantitative Bio Element Analysis and Mapping (QBEAM) Center, which is graciously supported by the Office of the Vice President for Research and Innovation, the Colleges of Natural Science, Human Medicine, Osteopathic Medicine, Veterinary Medicine, Engineering, Agriculture and Natural Resources with additional funding provided by the National Research Resource for Quantitative Mapping in the Life Sciences (QE Map) from the National Institute of General Medical Sciences (NIGMS) under grant P41GM135018. Author contributions Conceptualization: GR, KK. Methodology: MMC, GR. Investigation: MMC. Visualization: MMC, GR. Supervision: GR, KK. Writing—original draft: MMC, GR. Writing—review & editing: GR, MMC, KK. Competing interests Authors declare that they have no competing interests. Materials & Correspondence All correspondence and material requests should be addressed to Dr. Gemma Reguera ( [email protected] ). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8864095","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":594358618,"identity":"be652013-04cc-42e5-82b5-582da8480e0f","order_by":0,"name":"Gemma Reguera","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYBADOQjFRoIWY9K1JDYQrcW8vf2axMc9dulr+88YMHwoO0xYi8yZM2WSM54l5267kWPAOOMcEVokJHLSpHkOMAO18Bgw87YRo0X+TZr0nwP16Wbnzxgw/yVKiwT7MWmGA4cTzA7kGDAzEqWFJ4fZsufAccNtN9IKDvacSydCC/vxhzd+HKiWNzt/eOODH2XWhLUwMPAYwJkHiFEPBOwPiFQ4CkbBKBgFIxYAAKFYOsKS5JLcAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-4317-7933","institution":"Michigan State University","correspondingAuthor":true,"prefix":"","firstName":"Gemma","middleName":"","lastName":"Reguera","suffix":""},{"id":594358619,"identity":"f5cb3d41-dceb-4d9b-93fd-4d13b323b8f0","order_by":1,"name":"Morgen Clark","email":"","orcid":"","institution":"Michigan State University","correspondingAuthor":false,"prefix":"","firstName":"Morgen","middleName":"","lastName":"Clark","suffix":""},{"id":594358620,"identity":"13595256-0f94-4946-8046-a745990aa2eb","order_by":2,"name":"Kazem Kashefi","email":"","orcid":"","institution":"Michigan State University","correspondingAuthor":false,"prefix":"","firstName":"Kazem","middleName":"","lastName":"Kashefi","suffix":""}],"badges":[],"createdAt":"2026-02-12 16:16:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8864095/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8864095/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103386051,"identity":"d4b2e0fa-5c8b-44f5-b8bf-00ae3d31ec4a","added_by":"auto","created_at":"2026-02-25 06:42:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1782817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNa\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e ionic strength modulates vesiculation. (A-B) \u003c/strong\u003eAFM micrographs (\u003cstrong\u003eA\u003c/strong\u003e;\u003cstrong\u003e \u003c/strong\u003escale bar, 1 µm) used to quantitate OMV formation (\u003cstrong\u003eB; \u003c/strong\u003eper µm\u003csup\u003e2\u003c/sup\u003e and OD\u003csub\u003e600\u003c/sub\u003e of 0.5) in NB, DB and salt-amended DB (DB\u003csup\u003e+\u003c/sup\u003e and DB\u003csup\u003e++\u003c/sup\u003e) cultures. (\u003cstrong\u003eC\u003c/strong\u003e) Histograms showing average periplasmic widths in 8-10 cells imaged via cryo-electron microscopy (representative images shown below). \u003cstrong\u003e(D)\u003c/strong\u003e Cell size (AFM area) in NB, DB or salt-amended DB cultures (DB\u003csup\u003e+\u003c/sup\u003e, DB\u003csup\u003e++\u003c/sup\u003e) imaged in AFM scans such as those shown in (\u003cstrong\u003eA\u003c/strong\u003e). Statistical analyses were conducted with a two-tailed \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 in bold).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8864095/v1/d77815f6f3c4d298634f98fc.png"},{"id":103385910,"identity":"95f8cf8b-ff70-48ce-8409-5f78f6384a75","added_by":"auto","created_at":"2026-02-25 06:41:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1444909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNa\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-induced reprogramming of cell envelope proteins.\u003c/strong\u003e (\u003cstrong\u003eA-B\u003c/strong\u003e) Heat maps show differentially expressed proteins \u003cstrong\u003e(A) \u003c/strong\u003eand RNA transcripts \u003cstrong\u003e(B)\u003c/strong\u003e in DB vs. NB cultures, including outer membrane-cell wall anchors (arrows) and proteins involved in extracellular electron transfer (asterisks). One of the Pba values in the proteomic dataset could not be retrieved under the experimental conditions (NA, “not available”), and was imputed. \u003cstrong\u003e(C)\u003c/strong\u003e Model illustrating how cell wall distortions due to reduced linking outer membrane-cell wall anchoring to cell wall distortions at low ionic strengths and vesiculation. Abbreviations: amino acids in the peptide chain (L-Ala, \u003cem\u003eL\u003c/em\u003e-alanine 1; D-Glu, \u003cem\u003eD\u003c/em\u003e-glutamic acid 2; mDAP, meso-diaminopimelic acid 3; D-Ala, \u003cem\u003eD\u003c/em\u003e-alanine 4), peptidoglycan sugars (GlcNAc, \u003cem\u003eN\u003c/em\u003e-acetylglucosamine; MurNAc, \u003cem\u003eN\u003c/em\u003e-acetylmuramic acid); outer membrane-peptidoglycan anchors Lpp (lipoprotein trimer, adapted from the \u003cem\u003eE. coli\u003c/em\u003e crystal structure PDB: 1EQ7) and Pba (peptidoglycan-binding anchor, dimer representation); other abbreviations (IM, inner membrane; LPS, lipopolysaccharide; OM, outer membrane; OMV, outer membrane vesicle; PG, peptidoglycan).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8864095/v1/4332387b3da66f540974e85e.png"},{"id":103385982,"identity":"92aaa8fc-be0b-47dd-b91c-b95e61ab3582","added_by":"auto","created_at":"2026-02-25 06:42:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1280642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of uranium on envelope structure, vesiculation, and piliation.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Histograms showing average periplasmic widths in uranium-treated NB and DB cells (8-10 per condition) imaged via cryo-EM (representative images shown, with arrow pointing at protruding pili; scale, 50 nm). The violin plots show cell sizes measured as AFM area for each culture. \u003cstrong\u003e(B)\u003c/strong\u003e Representative AFM images of uranium-treated NB and DB cultures showing cells, OMVs, and monolateral mineralization of uranium by piliated cells. The relative vesiculation of the uranium-treated versus untreated cultures is also shown. (\u003cstrong\u003eC\u003c/strong\u003e) Correlation between vesiculation and piliation in the uranium-treated and untreated NB and DB cultures. Statistical analyses were conducted with a two-tailed \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 in bold).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8864095/v1/b44abdc3d93f2f9a1e076b18.png"},{"id":103385966,"identity":"4b269c74-36d9-40de-b97a-faff3bf679f7","added_by":"auto","created_at":"2026-02-25 06:42:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":315508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of uranyl cations on growth and detoxification. (A) \u003c/strong\u003eGrowth curves (optical density at 600 nm, OD\u003csub\u003e600\u003c/sub\u003e, of \u0026gt;6 biological replicates at 35\u003csup\u003eo\u003c/sup\u003eC) and efficiency indicators (lag phase, doubling time, and maximum growth yields as OD\u003csub\u003e600\u003c/sub\u003e and percentage of viable cells measured with the LIVE/DEAD\u003csup\u003e®\u003c/sup\u003e BacLight\u003csup\u003eTM\u003c/sup\u003e kit) in the absence (gray) or presence (ochre) of 500 mM uranyl acetate. \u003cstrong\u003e(B)\u003c/strong\u003e Resting (growth-arrested) cell suspension assays with mid-exponential cells from \u003cstrong\u003e(A)\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eTop panel shows viability (BacLight fluorescence dyes ratio) of resting cells from NB and DB cultures. Bottom panel shows average uranium removal (µM) per viable cell in triplicate biological replicates and two technical replicates after incubating (35\u003csup\u003eo\u003c/sup\u003eC) the resting cells in the presence of 500 µM uranyl acetate for 6 h. Statistical significance of treated versus untreated controls was assessed with a two-tailed \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.1 in bold) after a Grubb’s outlier test.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8864095/v1/4fd3f7f66593586476bceea9.png"},{"id":103386060,"identity":"34a454dd-c5cb-4e1c-a172-9f2851c905d9","added_by":"auto","created_at":"2026-02-25 06:42:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6108739,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8864095/v1/7e0d37ef-d6c7-43c0-bb00-e6d2c1702ab8.pdf"},{"id":103385953,"identity":"3e8c86f4-129b-456e-8d1e-67a934c5ee21","added_by":"auto","created_at":"2026-02-25 06:42:02","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":394272,"visible":true,"origin":"","legend":"\u003cp\u003eDataset S1\u003c/p\u003e","description":"","filename":"DataS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8864095/v1/8e0996d4e91ab1bb770f0c1c.xlsx"},{"id":103385947,"identity":"bae39b3d-389a-46df-a8e4-35fb780012cc","added_by":"auto","created_at":"2026-02-25 06:42:00","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":463688,"visible":true,"origin":"","legend":"\u003cp\u003eDataset S2\u003c/p\u003e","description":"","filename":"DataS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8864095/v1/54a5453bc37f9a4e315691a3.xlsx"},{"id":103385958,"identity":"afc9797f-de21-40e2-b08e-a0a4992f6639","added_by":"auto","created_at":"2026-02-25 06:42:03","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":24689360,"visible":true,"origin":"","legend":"Supplementary Information file","description":"","filename":"ClarkSuppMatNatComm.docx","url":"https://assets-eu.researchsquare.com/files/rs-8864095/v1/8e30d389c3e5b445e334a2e0.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Cation-driven envelope dynamics modulate outer membrane vesiculation and extracellular electron transfer in \u003ci\u003eGeobacter\u003c/i\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe biomineralization of iron and other metals by electrically active (electric) microorganisms represents one of the oldest and most influential geobiological processes on Earth\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. These microbes, and the conductive minerals they generate, establish natural electric grids that metabolically couple microbial communities and shape the geochemistry of entire ecosystems\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Among them, members of the \u003cem\u003eGeobacterales\u003c/em\u003e provide foundational models for understanding how microbial electrical networks drive metal\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and, indirectly, carbon\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e sequestration into stable mineral sinks and how their metabolisms can be harnessed to mine and reclaim metals critical to energy storage technologies\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. These electrical activities are primarily driven by a trans-envelope respiratory chain of \u003cem\u003ec-\u003c/em\u003ecytochromes\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e as well as retractable, conductive pili assembled on one side of the cell to extend the redox-active surface beyond outer membrane \u003cem\u003ec\u003c/em\u003e-cytochromes.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e The conductive pili shared many of the conserved properties of bacterial Type IVa pili, yet are assembled with a uniquely short,\u003csup\u003e11\u003c/sup\u003e aromatically dense,\u003csup\u003e13\u003c/sup\u003e and intrinsically conductive\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e pilin peptide. The ubiquity of similar, aromatically dense pilin sequences in metal-rich environments\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e suggests that pili-mediated electron flow is a widespread mechanism for metal cycling across ecosystems.\u003c/p\u003e \u003cp\u003ePilus nanowires also drive the reductive mineralization of soluble toxic metals and radionuclides such as the uranyl oxycation (UO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e).\u003csup\u003e16\u003c/sup\u003e Surface motifs of anionic ligands promote the binding of cationic metals to the pilus fibers to facilitate their reductive mineralization,\u003csup\u003e16\u003c/sup\u003e in a process that also conserves energy for growth.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Pilus nanowires also permeate the exopolysaccharide (EPS) matrix of biofilms, establishing vertical redox gradients with EPS-anchored \u003cem\u003ec\u003c/em\u003e-cytochromes for optimal electron flow\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and enhancing uranium mineralization.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Field amendments with electron donors such as acetate stimulate these activities and promote the in situ immobilization of uranium and other toxic metals in contaminated environments.\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Guiding these and other applications are laboratory studies with representative strains, primarily \u003cem\u003eGeobacter sulfurreducens\u003c/em\u003e, grown in bicarbonate-based mineral media formulated with bicarbonate and mineral salts that replicate the geochemistry of contaminated sites.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e These studies identified a complementary pathway for the detoxification of uranyl cations via their adsorption to the rough (no O-antigen) lipopolysaccharide (LPS) that densely coats \u003cem\u003eGeobacter\u003c/em\u003e cells and the disposal of LPS-bound metal in outer membrane vesicles (OMVs)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Vesiculation also provides a mechanism for the detoxification of any uranium which may have diffused and mineralized in the periplasm\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e due to the high-affinity and low-potential of periplasmic \u003cem\u003ec\u003c/em\u003e-cytochromes\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). The presence of \u003cem\u003ec\u003c/em\u003e-cytochromes in the OMVs also suggests roles in extracellular electron transfer to minerals and other cells in biofilms.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Furthermore, OMVs readily attach to solid surfaces and lower interfacial resistance.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Thus, the vesicles might complement or amplify mineralization reactions driven by the pili.\u003c/p\u003e \u003cp\u003eCultivation conditions strongly bias whether cells assemble pili or vesiculate, thus undermining the reproducibility and predictive power of laboratory studies. For examples, pilus nanowires are required for iron or manganese oxides respiratory growth but not for the respiration of soluble electron acceptors such as fumarate.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Yet, non-piliated cells can grow optimally in fumarate cultures under uranium stress by hypervesiculating.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Furthermore, vesiculation is attenuated in the fumarate-grown cultures at temperatures (25\u003csup\u003eo\u003c/sup\u003eC) that slow down growth as in cultures with metal oxides and induce pili assembly.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e These reciprocal responses suggest that pili-wired and OMV-based detoxification pathways are co-regulated. Evidence further implicates outer membrane-peptidoglycan crosslink rearrangements in this coordination. Genetic disruption of one of several outer membrane OmpA-like anchors in \u003cem\u003eG. sulfurreducens\u003c/em\u003e increases both vesiculation and current harvesting from electrode-associated biofilms.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Moreover, the hypervesiculating cells release OMVs enriched in OmcZ,\u003csup\u003e30\u003c/sup\u003e an outer membrane \u003cem\u003ec\u003c/em\u003e-cytochrome that accumulates closer to the electrode surface to lower interfacial resistance.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Yet as the biofilm cells grow away from the electrode, the wiring capacity of the conductive pili becomes increasingly more important\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, enhancing current harvesting\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and uranium detoxification.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e These observations point to a coordinated regulation of pili- and OMV-based electron transfer pathways via yet unresolved envelope mechanisms.\u003c/p\u003e \u003cp\u003eMotivated by this gap in knowledge, we investigated how \u003cem\u003eG. sulfurreducens\u003c/em\u003e modulates vesiculation when grown in two mineral media (NB and DB) indistinctively used for \u003cem\u003eGeobacter\u003c/em\u003e cultivation. NB, a nutrient broth originally formulated for genetic studies,\u003csup\u003e32\u003c/sup\u003e remains a popular medium for metal reduction and OMV research.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e DB is an NB-derived mineral formulation originally used for electrochemical studies of electrode-immobilized cells\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e used, once supplemented with the NB vitamin mix, for electrochemical\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and metal detoxification\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e growth studies. Both media use bicarbonate buffering to recreate the complexation of cationic metals in the contaminated subsurface.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Yet, as we show, previously overlooked differences in the media cationic strength, particularly from sodium (Na\u003csup\u003e+\u003c/sup\u003e), influence outer membrane-peptidoglycan crosslinks and reciprocally regulate piliation and vesiculation. These findings refine current models of OMV control in Gram-negative bacteria and reveal physicochemical drivers of envelope remodeling that ultimately determine the reproducibility of laboratory phenotypes and metal transformations driven by \u003cem\u003eGeobacter\u003c/em\u003e bacteria. We further discuss the implications of these findings for translating laboratory observations into field-scale and energy-harvesting applications with \u003cem\u003eGeobacter\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eControl of outer membrane vesiculation by Na\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eionic strength.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to standardize culture conditions for the study of vesiculation in \u003cem\u003eG. sulfurreducens\u003c/em\u003e, we quantified OMVs in mid-exponential acetate-fumarate cultures using two popular mineral media (NBAF\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and DBAF,\u003csup\u003e34\u003c/sup\u003e respectively, but abbreviated as NB and DB herein). Incubation was at 35\u003csup\u003eo\u003c/sup\u003eC to support optimal planktonic growth and prevent piliation.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e OMV detection leveraged the high adsorption capacity of highly-oriented pyrolytic graphite (HOPG) for extracellular vesicles and the imaging power of an atomic force microscope (AFM)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e to track vesiculation levels in culture samples, as previously described.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e The method also avoided the artefacts introduced with protocols requiring OMV extraction, purification, and single-particle tracking analysis,\u003csup\u003e37\u003c/sup\u003e previously applied to characterize \u003cem\u003eG. sulfurreducens\u003c/em\u003e OMVs.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Although the NB and DB formulations contained comparable levels of sodium bicarbonate (21.4 and 23.8 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, respectively) and were identically buffered (pH of 6.8) and amended with vitamins, their salt content differed subtly (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Most notably, the DB medium lacked two weakly complexed Na\u003csup\u003e+\u003c/sup\u003e salts (6 mM NaCl and 3.5 mM Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e) from the NB formulation, decreasing the molar concentration of the alkali cation in DB from 89.5 to 79.3 mM. This difference markedly influenced the vesiculation response, stimulating it in the DB cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) despite measurably impacts on outer membrane permeability (\u003cb\u003eFig. S2A\u003c/b\u003e). However, addition of one or two of the NB salts to the DB cultures chemically rescued the hypervesiculation phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Overall, average OMV yields per cell linearly scaled with the molar Na\u003csup\u003e+\u003c/sup\u003e concentration in the cultures (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.999).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther supporting the direct role of Na\u003csup\u003e+\u003c/sup\u003e in vesiculation control, OMV formation in the NB cultures was further attenuated by adding yeast extract (\u003cb\u003eFig. S3\u003c/b\u003e), a concentrate of the soluble fraction of osmotically autolyzed yeast that carries high molar concentrations of Na\u003csup\u003e+\u003c/sup\u003e salts\u003csup\u003e38\u003c/sup\u003e and is commonly added to \u003cem\u003eG. sulfurreducens\u003c/em\u003e cultures.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e By contrast, NB supplementation with cysteine, a reducing agent commonly added to ensure anoxic conditions,\u003csup\u003e32,40\u003c/sup\u003e had no effect on OMV release (\u003cb\u003eFig. S3\u003c/b\u003e). These results suggested that Na\u003csup\u003e+\u003c/sup\u003e ionic strength is a key modulator of outer membrane vesiculation in \u003cem\u003eG. sulfurreducens\u003c/em\u003e. Further supporting this, increasing the concentration of sodium acetate as an electron donor did not significantly impact vesiculation until the weakly complexed NB salts were added (\u003cb\u003eFig. S2\u003c/b\u003e), consistent with the stronger binding of Na\u003csup\u003e+\u003c/sup\u003e to acetate (CH\u003csub\u003e3\u003c/sub\u003eCOO⁻) than to chloride (Cl\u003csup\u003e\u0026ndash;\u003c/sup\u003e) or carbonate (CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026ndash;\u003c/sup\u003e) anions predicted from the Hofmeister series.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e By contrast, the type of alkaline ion mattered and higher concentrations of K\u003csup\u003e+\u003c/sup\u003e than Na\u003csup\u003e+\u003c/sup\u003e chloride and carbonate salts were needed to attenuate the hypervesiculation response of DB-grown cells to NB levels (\u003cb\u003eFig. S2\u003c/b\u003e). This is consistent with a mechanism for vesiculation control mediated by electrostatic interactions that sterically favored the smaller Na\u003csup\u003e+\u003c/sup\u003e cation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNa\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eionic strength remodels outer membrane-peptidoglycan crosslinks and respiratory chains.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhat type of electrostatic interactions with Na\u003csup\u003e+\u003c/sup\u003e could modulate OMV formation? This alkali monovalent cation, more so than K\u003csup\u003e+\u003c/sup\u003e, diffuses freely through the outer membrane of Gram-negative cells\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and strongly binds the peptidoglycan\u0026rsquo;s carboxylate groups,\u003csup\u003e43\u003c/sup\u003e minimizing repulsion forces that would otherwise cause cell wall expansions.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Consistent with this, cryo-EM imaging (\u003cb\u003eFig. S4\u003c/b\u003e) revealed enlarged periplasms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and, accordingly, larger cell surface area (measured by AFM, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) in DB-grown cells compared to those from NB or salt-amended DB cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Cell size also scaled linearly with OMV formation across all culture conditions (\u003cb\u003eFig. S5\u003c/b\u003e), strengthening the link between Na\u003csup\u003e+\u003c/sup\u003e-driven cell wall dynamics and vesiculation control.\u003c/p\u003e \u003cp\u003eWe gained further insights into envelope adaptations to Na\u003csup\u003e+\u003c/sup\u003e by comparing the proteomes (\u003cb\u003eData S1\u003c/b\u003e) and transcriptomes (\u003cb\u003eData S2\u003c/b\u003e) of DB-grown cells to NB controls. Though relatively modest (~\u0026thinsp;10 mM), differences in Na\u003csup\u003e+\u003c/sup\u003e molar concentrations in the two media drove broad changes in envelope-associated proteins (\u003cb\u003eFig. S6-7\u003c/b\u003e), including lipoproteins tethering the outer membrane to the underlying peptidoglycan (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The most significant change was the upregulation in the DB-grown cells of Lpp, the abundant Braun lipoprotein that covalently crosslinks the outer membrane to the peptidoglycan to preserve its integrity\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e and mechanically stabilize the cell envelope.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e The Lpp of \u003cem\u003eG. sulfurreducens\u003c/em\u003e is also longer than canonical Lpp proteins and well suited to accommodate periplasmic enlargements,\u003csup\u003e47\u003c/sup\u003e such as those observed in the DB-grown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Pba, one of several OmpA-like \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ep\u003c/span\u003eeptidoglycan-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eb\u003c/span\u003einding protein \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ea\u003c/span\u003enchors implicated in vesiculation control in \u003cem\u003eG. sulfurreducens\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and other Gram-negative bacteria,\u003csup\u003e48\u003c/sup\u003e was significantly downregulated in the transcriptomic dataset and, to a lesser extent, in the proteome (significance after imputation of a missing value was \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.12) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProteins of the Type IV pilus machinery (PilM) and the structural subunit of the conductive pili (PilA pilin)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e were also upregulated in the DB proteomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The trans-envelope pilus complex not only enhances the respiratory capacity of the cells,\u003csup\u003e11\u003c/sup\u003e it also reinforces outer membrane-peptidoglycan crosslinks \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. DB-grown cells also upregulated other proteins involved in extracellular electron transfer (\u003cb\u003eTable S2\u003c/b\u003e), most notably the XapD ABC transporter and XapK glycosyltransferase required for the synthesis of the redox-active Xap EPS\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e and the small PgcA \u003cem\u003ec\u003c/em\u003e-cytochrome that facilitates interfacial electron transfer with minerals\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Transcriptional profiling (\u003cb\u003eFig. S7\u003c/b\u003e) revealed additional reprogramming of membrane-bound proteins not captured within the significant thresholds set for the proteomic data (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cb\u003eTable S3\u003c/b\u003e). These proteins included outer membrane \u003cem\u003ec\u003c/em\u003e-cytochromes involved in iron oxide reduction such as OmcS\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e and those (OmcG and OmcH) involved in their regulation.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Many of these \u003cem\u003ec\u003c/em\u003e-cytochromes are enriched in OMVs\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and could have been underrepresented in the cells harvested for quantitative proteomics (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and heme-staining detection (\u003cb\u003eFig. S7\u003c/b\u003e). Heme-staining is further compounded by the similar electrophoretic mobility of many envelope \u003cem\u003ec\u003c/em\u003e-cytochromes,\u003csup\u003e54,55\u003c/sup\u003e which can mask expression changes. Similarly, transcriptomics but not proteomics analysis revealed the upregulation in DB-grown cells of the ATP-binding subunit of an ABC exporter (YbhF) with predicted roles in Na\u003csup\u003e+\u003c/sup\u003e:H\u003csup\u003e+\u003c/sup\u003e antiport\u003csup\u003e56\u003c/sup\u003e and the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ee\u003c/span\u003extra\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ec\u003c/span\u003eytoplasmic \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ef\u003c/span\u003eunction (ECF) sigma factor RpoE (\u003cb\u003eFig. S7\u003c/b\u003e), an alternative sigma factor that modulates the expression of pilus nanowires and \u003cem\u003ec\u003c/em\u003e-cytochromes in \u003cem\u003eG. sulfurreducens.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e The functional significance of these transcriptional shifts in cation-driven envelope remodeling remains to be determined. Notably, RpoE regulates OMV production and other envelope responses in numerous Gram-negative bacteria,\u003csup\u003e58\u003c/sup\u003e yet is role in \u003cem\u003eG. sulfurreducens\u003c/em\u003e is largely unexplored. This highlights the need for further studies to determine how this bacterium reprograms envelope responses to cation-driven structural adaptations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEnvelope adaptations to the uranyl oxycation reveal dynamic trade-offs between vesiculation and piliation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eLike Na\u003csup\u003e+\u003c/sup\u003e, the uranyl oxycation also strongly binds and effectively neutralizes the peptidoglycan carboxylates.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e Consistent with this, culture supplementation with uranyl acetate (DB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e and NB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e) abolished differences in the periplasmic width and surface area of the DB- versus NB-grown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The average periplasmic width of uranium-treated cells in both media was among the largest measured (~\u0026thinsp;30\u0026ndash;32 nm), as expected from the complexation of the peptidoglycan carboxylates with the much larger uranyl oxycation. However, the average size of uranium-treated cells was smaller than in untreated cultures (\u003cb\u003eFig. S8A\u003c/b\u003e), suggesting that peptidoglycan complexation with the rigid, linear uranyl oxycation (O\u0026thinsp;=\u0026thinsp;U=O\u003csup\u003e2+\u003c/sup\u003e) imposed constrains to overall cell expansions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUranium supplementation also triggered piliation in both cultures, albeit more strongly in the DB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e than in the NB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e cultures (14.4% of the cells had extensive mineralization compared to 8.5% in NB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C). AFM scans showed the characteristic monolateral mineralization of uranium by piliated cells\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e in the cultures and identified, at closer magnification, uranium nanoparticles permeated by pilus fibers with the characteristic AFM height (2.5 nm) of uranium-reducing pili\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eFig. S9\u003c/b\u003e). Furthermore, uranium-treated cells agglutinated (\u003cb\u003eFig. S8B\u003c/b\u003e), an aggregative phenotype associated with pili production.\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e Thus, uranyl cations not only counteracted the structural effects of Na⁺ ionic strength but also shifted the cellular response toward enhanced piliation and mineralization. AFM scans of uranium-treated cultures revealed OMVs with a similar AFM diameter (~\u0026thinsp;10 nm) as in the untreated cultures (\u003cb\u003eFig. S9\u003c/b\u003e). However, vesiculation yields were reversed in uranium-treated cultures, decreasing in DB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e and increasing in NB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, OMV release per cell inversely correlated with the levels of piliated cells in the uranium-treated cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These findings suggest dynamic trade-offs between pili and OMV pathways during uranium detoxification, where ionic conditions favoring piliation attenuate vesiculation and vice versa. Yet both mechanisms are needed to protect the cell from uranyl toxicity. This is because pilus nanowires confine mineralization to a single side of the cell,\u003csup\u003e11,16\u003c/sup\u003e leaving the opposite side reliant on uranyl binding to its rough LPS layer and detoxification via OMV release.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferential impact of Na\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eand uranyl cations on growth and metal mineralization.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRigorous control of media preparation and culture passage ensured phenotypic reproducibility and unmasked subtle, yet significant, differences in growth efficiency as a function of cation chemistry (Na\u003csup\u003e+\u003c/sup\u003e and uranyl) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The DB-grown cells reproducibly underwent slightly longer (\u0026gt;\u0026thinsp;1 hour) lag phases yet, once acclimated, they doubled faster than in the NB medium. Moreover, growth yields measured by optical density were similar in both cultures but masked the lower viability of the DB cells upon entry into stationary phase. Mid-exponential DB cells also experienced more viability losses upon growth arrest in a bicarbonate-buffered solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Additionally, the enzymatic removal of uranyl acetate per viable cell in the resting state was higher when using DB-grown cells compared to NB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The upregulation in the DB cells of envelope proteins for extracellular electron transfer such as pili and \u003cem\u003ec-\u003c/em\u003ecytochromes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) could have accounted for these differences, as previously shown.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Cell wall and periplasmic expansions in the DB-grown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) may have also made more peptidoglycan carboxylates available for binding the uranyl cations and prime them for more efficient uranium removal once in the resting state. Ultimately, these results caution about the importance of cultivation conditions in the metal-reducing phenotypes reported in resting cell assays, whose reproducibility depends on respiratory capacity\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e and cellular integrity.\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also compared the growth efficiency of \u003cem\u003eG. sulfurreducens\u003c/em\u003e in NB or DB cultures supplemented with 500 \u0026micro;M uranyl acetate (NB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e and DB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e cultures) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The NB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e and DB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e cultures acclimated and grew at comparable rates in the presence of uranium, albeit less efficiently than in untreated controls (\u003cb\u003eFig. S10\u003c/b\u003e). However, growth yields (both by optical density and viable cell counts) were significantly lower in the DB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e medium compared to NB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The heightened susceptibility of growth-arrested DB cells to uranium likely reflects the compounded effects of uranium-induced envelope stress on cells already compromised by low Na⁺ ionic strength, which makes their envelope more vulnerable to uranium toxicity once growth is arrested and active mechanisms for detoxification and repair are no longer active.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFindings from this work demonstrate that cation chemistry is a key and previously underappreciated regulator of outer‑membrane vesiculation and envelope remodeling in Gram-negative bacteria. Subtle, yet physiologically relevant, differences in the medium\u0026rsquo;s Na⁺ ionic strength remodeled outer membrane-peptidoglycan crosslinks, adjusted the periplasmic volume and cell size, and modulated vesiculation in \u003cem\u003eG. sulfurreducens\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These adaptive responses to Na\u003csup\u003e+\u003c/sup\u003e ionic strength reflect the free diffusion of the alkali cation through the outer membrane,\u003csup\u003e42\u003c/sup\u003e its strong affinity for peptidoglycan carboxylates,\u003csup\u003e43\u003c/sup\u003e and its ability to neutralize carboxylate charges to prevent repulsion forces that would otherwise cause cell wall expansions.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Insufficient free Na\u003csup\u003e+\u003c/sup\u003e in the DB medium led to cell wall expansions that widened the periplasm and destabilized non-covalent tethering via Pba, inducing curvature stress and vesiculation, even though the outer membrane-peptidoglycan lattice was covalently reinforced with Lpp anchors (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, culture supplementation with weakly complexed Na\u003csup\u003e+\u003c/sup\u003e salts alleviated these constraints and chemically rescued the hypervesiculating phenotype of DB-grown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). K\u003csup\u003e+\u003c/sup\u003e similarly modulated OMV formation but less effectively than Na\u003csup\u003e+\u003c/sup\u003e due its larger ionic radius and lower ionization potential,\u003csup\u003e63\u003c/sup\u003e which makes local ion pairing with peptidoglycan carboxylate ligands more inefficient.\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e These results highlight how steric and electrostatic factors unique to each alkali metal ion contribute to envelope remodeling. Conversely, the inability of additional sodium acetate to suppress vesiculation in DB cultures (\u003cb\u003eFig. S2\u003c/b\u003e) underscores how anion pairing can strongly limit free Na⁺ and cation-driven envelope adaptations.\u003c/p\u003e \u003cp\u003eControl of vesiculation via cation-driven remodeling of envelope crosslinks complements genetic studies linking outer membrane-peptidoglycan tethers to OMV formation, particularly those mediated by non-covalent OmpA-like anchors such as Pba\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e and the Braun lipoprotein Lpp.\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e Importantly, because vesiculation control in \u003cem\u003eG. sulfurreducens\u003c/em\u003e only required subtle changes in cation chemistry, we circumvented the broad pleiotropic defects reported for anchor mutants\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e and instead examined envelope adaptations under physiological relevant conditions. As a mechanically tethered system,\u003csup\u003e66\u003c/sup\u003e the outer membrane-cell wall lattice synchronously responds to cation deficiencies via peptidoglycan expansions that widen the periplasm.\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eG. sulfurreducens\u003c/em\u003e adapted to the periplasmic enlargements in DB cultures by making more Lpp (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), a trimeric anchor that covalently reinforces outer membrane-peptidoglycan crosslinks to stabilize the envelope.\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e By transitioning from tilted to stretched triple coiled-coil configurations, the flexible Lpp trimer stabilizes the envelope and preserves the integrity of the outer membrane during periplasmic enlargements.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e This capacity is further enhanced in \u003cem\u003eGeobacter\u003c/em\u003e due to its unusually long Lpp.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e Studies also show that the tilted-to-stretched transition of the Lpp trimer during periplasmic enlargements quickly dislodges non-covalent Pba anchors,\u003csup\u003e47\u003c/sup\u003e leaving untethered areas more prone to vesiculation and producing undulating membrane features, the same phenotypes we observed in the DB-grown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The reciprocal regulation of Pba and Lpp anchors likely arises as a compensatory response to the structural remodeling of cross-links triggered by low ionic strength. The possibility of fine-tuning these crosslinks and regulatory feedbacks via adjustments in the culture\u0026rsquo;s Na\u003csup\u003e+\u003c/sup\u003e ionic strength makes \u003cem\u003eGeobacter\u003c/em\u003e a powerful system to study the plasticity of the Gram-negative envelope to spatial constraints imposed by chemical cues.\u003c/p\u003e \u003cp\u003eLike Na\u003csup\u003e+\u003c/sup\u003e, the uranyl oxycation (UO₂\u0026sup2;⁺) exhibits a strong affinity for peptidoglycan carboxylate groups,\u003csup\u003e43\u003c/sup\u003e effectively neutralizing their negative charges and counteracting the differential effects of Na\u003csup\u003e+\u003c/sup\u003e ionic strength on periplasmic and cell surface area expansions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Nevertheless, uranium imposes additional physicochemical constraints on the envelope due its larger size, rigid linear structure (O\u0026thinsp;=\u0026thinsp;U=O\u003csup\u003e2+\u003c/sup\u003e), strong bidentate bonds, and ability to form polynuclear U(VI) complexes with carboxylates that yield a rigid and expanded uranium-peptidoglycan scaffold.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e Consequently, uranium-treated cells had, on average, larger periplasms than untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Yet their cell size was constrained by the rigidity of the cell wall (\u003cb\u003eFig. S8\u003c/b\u003e). These physicochemical properties also promoted robust assembly of the Type IV pilus machinery for the reductive mineralization of uranium (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The slightly enlarged periplasms of the uranium-treated cells likely alleviated spatial barriers to the orderly assembly and alignment of pre-pilus intermediates across the \u003cem\u003eGeobacter\u003c/em\u003e multi-layered envelope,\u003csup\u003e71\u003c/sup\u003e whereas the more rigid peptidoglycan scaffold provided mechanical support to the peptidoglycan anchors of the pilus complex.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e As a result, \u003cem\u003eG. sulfurreducens\u003c/em\u003e pili can more effectively counteract the destabilizing forces imposed by the antagonistic cycles of pilus protrusion and retraction that are key to effective uranium mineralization and respiratory gains.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e However, DB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e cultivation, more so than NB\u003csup\u003e+\u0026thinsp;U\u003c/sup\u003e, markedly increased piliation, consistent with compounding effects of peptidoglycan-binding cations on envelope remodeling. However, cation‑driven changes in the envelope imposed a dynamic tradeoff between pili‑mediated mineralization and OMV‑mediated export, reciprocally regulating the two pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This balance likely reflects the compounding effects of steric crowding and stronger tethering of the outer membrane in piliated cells, which spatially and structurally limits OMV biogenesis. Such constrains reciprocally coordinate the selection of pathways during metal detoxification, preventing envelope stress yet having opposing impacts on respiratory gains and metal transformations (pili-mediated mineralization versus reversible adsorption to vesicles).\u003c/p\u003e \u003cp\u003eTaken together, our results demonstrate that cation chemistry is an unappreciated experimental variable that must be rigorously controlled to ensure reproducibility in studies of extracellular electron transfer and metal transformations. Many discrepancies in the \u003cem\u003eGeobacter\u003c/em\u003e literature, and likely in other Gram‑negative systems, might be traced to unstandardized cations (e.g., Na⁺, K⁺) and associated counterions in media formulations and, potentially, other cultivation variables modulating piliation such as temperature.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Genetic analyses in \u003cem\u003eGeobacter\u003c/em\u003e further confound experimental results across labs due to the pleiotropic nature of most disruptions of envelope proteins, particularly pili and outer‑membrane \u003cem\u003ec\u003c/em\u003e-cytochromes.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e In contrast, chemical modulation of envelope mechanics provides a powerful and physiologically relevant tool to dissect these pathways without exacerbating compensatory responses. The compounding effects of cation-driven envelope adaptations on bioremediation outcomes and the performance of energy-harvesting technologies driven by \u003cem\u003eGeobacter\u003c/em\u003e are yet to be addressed. Moreover, the diverse and poorly understood repertoire of OmpA-like anchors in \u003cem\u003eG. sulfurreducens\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e represents an opportunity to dissect how chemical cues selectively bias envelope crosslinks toward piliation or vesiculation. Given the roles of OMVs in transporting \u003cem\u003ec\u003c/em\u003e-type cytochromes and modulating interfacial electron transfer reactions in \u003cem\u003eGeobacter\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and their ability to form conductive nanotubes in other bacteria\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e accounting for local ionic conditions will prove essential for producing reproducible datasets that can meaningfully guide bioremediation and energy systems driven by \u003cem\u003eGeobacter\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eBacterial strains and culture conditions.\u003c/b\u003e \u003cem\u003eG. sulfurreducens\u003c/em\u003e PCA\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e and ∆\u003cem\u003eomcS\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e and ∆\u003cem\u003eomcB\u003c/em\u003e mutants\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e from our culture collection were routinely grown at 35\u003csup\u003eo\u003c/sup\u003eC in NBAFYE\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, a nutrient broth (NB) medium with acetate (15 mM), fumarate (40 mM), cysteine (1 mM) and yeast extract (0.1%, w/v). After three transfers (starting OD\u003csub\u003e600\u003c/sub\u003e of 0.01\u0026ndash;0.03) in mid-exponential phase (0.4\u0026ndash;0.6 OD\u003csub\u003e600\u003c/sub\u003e), the cells were inoculated in test media: NBAF (no cysteine or yeast extract) or a modified media formulation DBAF.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e When indicated, cultures were amended with various salts (NaCl, Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, KCl, and/or potassium phosphate salts) or with 500 \u0026micro;M uranyl acetate. Growth parameters (length of lag phase, generation time, and maximum growth yields) were calculated using the growthcurver package (v0.3.1) in R. Pili-mediated agglutination in the cultures was measured as the OD\u003csub\u003e600\u003c/sub\u003e difference before and after gentle tube inversion.\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eMicroscopy.\u003c/b\u003e Mid-exponential cultures were routinely imaged in tapping mode at 0.3 Hz with an Asylum Research Cypher S Atomic Force Microscope (AFM) system equipped with an AC240TS tip to count OMVs\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and uranium-mineralizing cells expressing pili (AFM amplitude images analyzed with the multi-point tool in ImageJ). OMV and pilus filament diameters were measured as AFM height with the Igor Pro (6.38B01) and Asylum AFM software (v16.31.232). The area of the cells in AFM Zsensor images was measured with the \u0026ldquo;analyze particles\u0026rdquo; tool of ImageJ, as described in the Supplementary materials.\u003c/p\u003e \u003cp\u003eCryo-EM imaging used a Thermo Scientific\u0026trade; Talos\u0026trade; Arctica\u0026trade; instrument outfitted with a Falcon 3 Direct Detection Device and a Ceta CCD camera and operated at 200 kV. Mid-exponential cell suspensions concentrated via centrifugation (5000x\u003cem\u003eg\u003c/em\u003e for 10 min) were deposited (3\u0026ndash;5 \u0026micro;l) on 300 copper mesh grids coated with a Quantifoil\u0026trade; R 3.5/1.0 carbon film (45 sec of negative glow discharge with a Ted Pella Pelco easiGlow), blotted (2.5 and 10 sec blot and wait times, respectively; blot force, -2; humidity, 100%; and temperature, 4\u0026deg;C), and vitrified in liquid ethane in a Vitrobot Mark IV system (ThermoFisher Scientific). Image acquisition used the EPU (Thermo Scientific) software in linear mode at a 22,000x magnification (pixel size 6.6 \u0026Aring;) with a defocus range of \u0026minus;\u0026thinsp;10 \u0026micro;m and a dose rate of 3.3\u0026nbsp;e/\u0026Aring;\u003csup\u003e2\u003c/sup\u003e/s for ~\u0026thinsp;2 sec. Periplasm widths were measured in ImageJ (\u0026ldquo;Analyze\u0026gt;Measure\u0026rdquo; function) by fitting a rotated rectangle (~\u0026thinsp;5 nm wide) on the envelope every 15\u0026ndash;20 nm, excluding the mid-center and poles (each ~\u0026thinsp;1/5th of the cell length).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFluorescence assays of outer membrane permeability and cell viability.\u003c/b\u003e Mid-exponential cells were harvested by centrifugation (8000x\u003cem\u003eg\u003c/em\u003e, 7 min), washed twice (5 mM HEPES-5 mM glucose buffer pH 7.2), and resuspended in \u0026frac12; vol of the same buffer before dispensing 100 \u0026micro;l of sample and buffer controls (blanks) in triplicate wells of a white-walled, clear-bottom 96-well plate (Alkali Scientific). A stock of 500 M N-Phenyl-1-naphthylamine (NPN) (Sigma-Aldrich) in acetone was diluted to 20 \u0026micro;M in assay buffer before adding 100 \u0026micro;l to each well, pipetting to mix, and incubating in the dark (room temperature, 10 min) before measuring (350 nm excitation, 420 nm emission) fluorescence from the hydrophobic fluorescent NPN probe after intercalation into the outer membrane phospholipids.\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCell viability was assayed with the Live/Dead\u0026reg; BacLight\u0026trade; Bacterial Viability Kit (ThermoFisher Scientific) in cells harvested by centrifugation (5,000x\u003cem\u003eg\u003c/em\u003e for 10 min) from 0.5-1 ml early-stationary phase cultures or resting cell suspensions, washed twice in 0.85% NaCl, and dispensed (100 \u0026micro;l) in triplicate wells of a 96-well plate (Wuxi NEST), avoiding peripheral wells (rows A and H, columns 1 and 12) for improved reproducibility. A 2X staining solution (4 \u0026micro;l of 5 mM Syto9 and 6 \u0026micro;l of 20 mM propidium iodide per 2 ml nuclease-free water) was added to each sample, pipetting to mix, before dark incubation (room temperature, 15 min) and fluorescence measurements in the green (485/530nm excitation/emission) and red (485/630nm excitation/emission) channels. The ratio of green over red fluorescence was used as a proxy of cell viability.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResting cell assays with uranium.\u003c/b\u003e Resting cell suspensions (OD\u003csub\u003e600\u003c/sub\u003e 0.1) used mid-exponential cells osmotically adjusted in sequential steps to maximize viability, as previously described\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e except that the suspensions were prepared in 20 ml of buffer with 500 \u0026micro;M of uranyl acetate. Samples collected before or after 6-h incubation (35\u003csup\u003eo\u003c/sup\u003eC) were filter-sterilized (0.22 \u0026micro;m), acidified in 0.8 N nitric acid, and diluted 200-fold in 3% nitric acid before measuring the uranium concentration in an Agilent 8900 triple quadrupole tandem inductively coupled proton mass spectrometer (QQQ-ICP-MS).\u003c/p\u003e \u003cp\u003e \u003cb\u003eProteomics and RNA-Seq analysis.\u003c/b\u003e Proteins and RNA transcripts extracted from triplicate 10-ml, mid-exponential NBAF and DBAF cultures were sequenced at Michigan State University Research Technology Support Facility (RTSF) and the SeqCenter (Pittsburg, PA), respectively, following standardized protocols. Protein extraction was from cells harvested by centrifugation (3,220x\u003cem\u003eg\u003c/em\u003e, 20 min) and washed three times in 10 mM Tris-HCl buffer (pH 7.7) before resuspending in 500 \u0026micro;l of buffer and centrifuging again in 2-ml cryotubes (8,000x\u003cem\u003eg\u003c/em\u003e, 10 min) to obtain a pellet for storage at -80\u003csup\u003eo\u003c/sup\u003eC. Cell lysis was initiated in 2x SDS-PAGE sample buffer (125 mM Tris, pH 6.8, 4% SDS, 20% glycerol, 50 mM DTT; 95\u003csup\u003eo\u003c/sup\u003eC for 5 min) and, after cooling to room temperature, continued in a water bath sonicator (3 cycles of 1 min sonication and 1 min rest). Protein (100 \u0026micro;g, measured with Pierce BCA assay) was digested with trypsin/LysC (Promega) using S-traps (ProtiFi) according to manufacturer\u0026rsquo;s instructions before resuspending 20 \u0026micro;l of the digest in 2% acetonitrile/0.1% trifluoroacetic acid and calculating the final peptide concentration with a Pierce Quantitative Colorimetric Peptide Assay Kit. Samples were then run through Data-Independent Acquisition (DIA) LC/MS/MS to obtain the peptide spectra and analyzed as described in the Supplementary Methods and elsewhere.\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e Missing (\u0026lsquo;NA\u0026rsquo;) values were separately imputed in NB and DB replicates with the R studio missForest package (v1.5).\u003csup\u003e80\u003c/sup\u003e Differentially expressed proteins (DEPs) (\u003cb\u003eData S1\u003c/b\u003e) were determined in Microsoft Excel, as described in the Supplementary Methods.\u003c/p\u003e \u003cp\u003eRNA extraction and purification was as previously described.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e Illumina library preparation and sequencing (paired end 150bp reads on a NovaSeq X Plus instrument) used protocols standardized at SeqCenter. Demultiplexing, quality control, and adapter trimming used Illumina\u0026rsquo;s bcl-convert (v4.2.4) software, whereas read mapping used HISAT2 (v2.2.0) with default parameters and \u0026rsquo;--very-sensitive\u0026rsquo;.\u003csup\u003e81\u003c/sup\u003e Read quantification used Subread\u0026rsquo;s featureCounts (v2.0.1) functionality with default parameters and \u0026lsquo;-Q20\u0026rsquo;.\u003csup\u003e82\u003c/sup\u003e Read counts loaded into R (v4.0.2) were normalized using edgeR\u0026rsquo;s (v1.14.5) Trimmed Mean of M values (TMM) algorithm with default parameters\u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e before conversion to counts per million (CPM). Differential expression analysis used edgeR\u0026rsquo;s glmQLFTest. The data was then filtered for a |log\u003csub\u003e2\u003c/sub\u003e FC| \u0026gt; 0.58 and \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 to determine differentially expressed genes (DEGs) (\u003cb\u003eData S2\u003c/b\u003e). Adjusted \u003cem\u003ep\u003c/em\u003e-values were not used given the low number of DEGs.\u003c/p\u003e \u003cp\u003eFunctional assignments to DEPs and DEGs used the KEGG and UniProt \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e databases. Analysis of transmembrane regions and subcellular localization was via DeepTMHHM (v1.0) and PSORTb (v3.0.3), respectively. Data visualization (heatmaps, dispersion plots, and volcano plots) used the pheatmap (v1.0.12) and ggplot2 (v3.5.1) packages in R studio.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical Analysis.\u003c/b\u003e The statistical significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of phenotypic differences in the various cultures was assessed with either a Student\u0026rsquo;s or Welch\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test (chosen based on the results of a F-test for variance) in Excel, as described in the corresponding figure legends. Where indicated, we used the Grubbs\u0026rsquo; test for outliers from GraphPad\u0026rsquo;s online outlier calculator to remove up to one outlier per condition.\u003c/p\u003e \u003cp\u003e \u003cb\u003eData and materials availability.\u003c/b\u003e All data are available in the main text or the supplementary materials. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD069850. The RNA-Seq raw data are available at the NCBI Gene Expression Omnibus (GEO) repository under accession number GSE285971.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available in the main text or the supplementary materials. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD069850. The RNA-Seq raw data are available at the NCBI Gene Expression Omnibus (GEO) repository under accession number GSE285971.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by U.S. Army Research Office grant 80459BB to GR and KK) and by National Science Foundation Emerging Frontiers in Research \u0026amp; Innovation grant 2318057 to GR.\u0026nbsp;We are grateful to Dr. Thomas J. Silhavy at Princeton University for discussions of preliminary data inspiring this research and to various colleagues at Michigan State University for technical support in various areas (Dr. Marcela Tabares, RNA-Seq; Nicholas Tefft and Dr. Michaela TerAvest, heme-stained gels; Dr. Sundharraman Subramanian, cryo-EM; and Douglas Whitten, proteomics). We also acknowledge Michigan State University Quantitative Bio Element Analysis and Mapping (QBEAM) Center, which is graciously supported by the Office of the Vice President for Research and Innovation, the Colleges of Natural Science, Human Medicine, Osteopathic Medicine, Veterinary Medicine, Engineering, Agriculture and Natural Resources with additional funding provided by the National Research Resource for Quantitative Mapping in the Life Sciences (QE Map) from the National Institute of General Medical Sciences (NIGMS) under grant P41GM135018.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: GR, KK. Methodology: MMC, GR. Investigation: MMC. Visualization: MMC, GR. Supervision: GR, KK. Writing\u0026mdash;original draft: MMC, GR. Writing\u0026mdash;review \u0026amp; editing: GR, MMC, KK.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials \u0026amp; Correspondence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll correspondence and material requests should be addressed to Dr. Gemma Reguera ([email protected]). \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eReguera G, Kashefi K (2019) The electrifying physiology of \u003cem\u003eGeobacter\u003c/em\u003e bacteria, 30 years on. Adv Microb Physiol 74:1\u0026ndash;96\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVargas M, Kashefi K, Blunt-Harris EL, Lovley DR (1998) Microbiological evidence for Fe(III) reduction on early Earth. 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Sci Adv 11:eadw4289\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBose A, Gardel EJ, Vidoudez C, Parra EA, Girguis PR (2014) Electron uptake by iron-oxidizing phototrophic bacteria. Nat Commun 5:3391\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao C et al (2024) The dual roles of dissimilatory iron reduction in the carbon cycle: The iron mesh effect can increase inorganic carbon sequestration. Glob Change Biol 30:e17239\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReguera G (2018) Harnessing the power of microbial nanowires. Microb Biotechnol 11:979\u0026ndash;994\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUeki T (2021) Cytochromes in extracellular electron transfer in \u003cem\u003eGeobacter\u003c/em\u003e. Appl Environ Microbiol 87:e03109\u0026ndash;e03120\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReguera G et al (2005) Extracellular electron transfer via microbial nanowires. 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Nucleic Acids Res 51:D523\u0026ndash;D531\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Outer membrane vesicles, peptidoglycan crosslinks, Gram-negative envelope, microbial nanowires, Type IV pili","lastPublishedDoi":"10.21203/rs.3.rs-8864095/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8864095/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eGeobacter\u003c/em\u003e bacteria use conductive pili and redox-active outer membrane vesicles to mediate metal transformations critical to the effectiveness of bioremediation and energy technologies. Mechanistic knowledge into these processes primarily comes from studies with \u003cem\u003eGeobacter sulfurreducens\u003c/em\u003e grown in media closely formulated to mirror the mineral chemistry of contaminated sites. Although subtle differences in the media\u0026rsquo;s cationic strength did not measurably change permeability, they reprogrammed outer membrane-peptidoglycan crosslinks modulating vesiculation and envelope functions impacting growth efficiency and mineralization. Cations that strongly bind and neutralize peptidoglycan carboxylates to prevent cell wall distortions, such as sodium and uranyl ions, ultimately determined the extent of envelope remodeling and cell bias toward pili-driven mineralization or membrane adsorption and release in vesicles. These findings identify cation chemistry as a key regulator of outer membrane vesiculation and the reprogramming of envelope functions ultimately determining the reproducibility of laboratory studies and effectiveness of bioremediation and energy-harvesting applications.\u003c/p\u003e","manuscriptTitle":"Cation-driven envelope dynamics modulate outer membrane vesiculation and extracellular electron transfer in Geobacter","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-25 06:40:13","doi":"10.21203/rs.3.rs-8864095/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dc778b3a-26df-40f6-9960-cd0b017b15f2","owner":[],"postedDate":"February 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63251253,"name":"Biological sciences/Microbiology/Bacteria/Bacterial physiology"},{"id":63251254,"name":"Earth and environmental sciences/Biogeochemistry/Element cycles"}],"tags":[],"updatedAt":"2026-04-08T13:57:52+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-25 06:40:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8864095","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8864095","identity":"rs-8864095","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}