Biology subjects

Ajo-Franklin, C. M.

Publications and source records attributed to Ajo-Franklin, C. M..

12 recordsLinked to original sources

bioRxiv2026

A Computational Framework for Domain Insertion into Type IV Pili for Bacterial Display and Living Material Assembly

Bacterial surface structures have enabled display systems with broad impact across biotechnology, but their narrow host range limits their deployment into diverse species. Conversely, Type IV Pili (TFP) are ubiquitous, structurally conserved appendages found across bacteria, but have been minimally explored for display. Here, we describe a computational framework for predicting viable insertion sites in major pilins for stable TFP-mediated display, which we apply to the major pilin PilA1 of the cyanobacterium Synechocystis sp. PCC 6803 to enable covalent binding to living materials. By analyzing an Alphafold3-generated PilA1 monomer alongside known multimeric TFP multimers, our pipeline identifies non-interfacial, solvent-accessible, and flexible sites for optimal PilA1 display. We probe these sites with both full-length and truncated SpyCatcher003 at two different expression levels. We show that cells expressing these PilA1-SpyCatcher003 fusions maintain up to 8-fold higher levels of cell suspension than previous C-terminal PilA1 display platforms, suggesting improved TFP assembly despite more than a two-fold increase in cargo size. Additionally, we validate SpyCatcher003 reactivity across the engineered strains, enabling covalent attachment of SpyTag003-containing proteins on the Synechocystis surface. Lastly, we utilize this covalent patterning to achieve a four-fold increase in Synechocystis loading into a living material without compromising its viscoelastic or mechanical properties. Taken together, this work provides a predictive framework for TFP engineering, and opens the door towards programmable surface display across the breadth of bacterial species.

synthetic biology↗

bioRxiv2026

Electrode Reduction by Vibrio natriegens Depends on Balanced Expression of Multiheme Cytochromes

Multiheme cytochromes c can facilitate electron transfer across the periplasm and outer membrane of Gram-negative bacteria to enable extracellular electron transfer (EET). EET empowers bacteria to maintain redox balance in oxygen-poor environments by donating electrons to solid materials. The resulting electron flux makes EET pathways useful tools for interfacing microorganisms with electronics. Recently, Vibrio natriegens, a marine bacterium notable for its rapid growth and expanding biotechnological applications, was found to perform iron reduction in a multiheme cytochrome c-dependent manner. However, the role of the V. natriegens EET genes in facilitating reduction of electrodes remains unexplored. Through single gene deletion and complementation, we find that each of cymA, pdsA, mtrA, and mtrB are required for production of electrical current by V. natriegens cultures. Curiously, deletion of the outer membrane decaheme cytochrome mtrC diminished but did not abolish electrode reduction. Modulating the induction of expression of mtrA and mtrC revealed that only a narrow range of induction of these decaheme cytochromes allows balanced cytochrome c production and EET. These findings indicate that a multiheme cytochrome-based EET pathway enables V. natriegens to reduce electrodes and that this pathway requires carefully balanced gene expression to function. This characterization of the role of multiheme cytochromes in the electroactivity of an emerging microbial chassis for biotechnology will enable new bioelectronic applications for V. natriegens and new understanding of the metabolic function of EET.

microbiology↗

bioRxiv2025

Mechanisms of soil carbon preservation illuminated by model mineral-associated organic matter

A central control on atmospheric CO2 is the stabilization of organic carbon in soils. While there are well-described properties associated with stable soil carbon, such as interactions with minerals and conserved chemical features, determining the mechanisms underlying these properties is challenged by soil complexity and heterogeneity. Here we synthesize a naturally complex, tunable model form of mineral-associated organic matter (LabMAOM) to explore mechanisms of stable soil carbon formation and persistence. We show that mineral association and ambient organic matter synergistically stabilize LabMAOM against microbial decomposition. Moreover, different starting compositions of organic matter yield a conserved final LabMAOM chemical composition consistent with natural soils. Our findings open new mechanistic understanding of stable soil carbon and will be of utility to climate modeling and soil health management.

synthetic biology↗

bioRxiv2025

Synthetic microbial co-cultures for modular bioelectronic sensing in diverse environments

Human disruption of ecosystems poses a significant threat to global health, driving the need for low-cost, low-power, and easily deployable sensors for environmental and health monitoring. Microbial bioelectronic sensors are particularly well-suited as they generate electrical signals and can be integrated into compact electronic devices for field deployment over extended periods. However, current engineering strategies for bioelectronic sensors lack modularity, are limited to a few microbial chassis, and depend on specialized instruments for signal detection. Here, we present the electroactive co-culture sensing system (e-COSENS), a plug-and-play platform for bioelectronic sensor development. This system comprises a "sender" bacterium that produces electron mediators in response to analytes and a "receiver" bacterium that utilizes the electron mediators to generate electrical signals via extracellular electron transfer (EET). By modularly swapping the sender bacterium and its associated genetic sensing elements, we achieved bioelectronic sensing of metals, small molecules, and peptides in distinct environmental, food, and human-relevant settings. Moreover, we designed a centimeter-sized bioelectronic device that enables low-cost, portable signal readout from e-COSENS using a household digital multimeter. The e-COSENS platform greatly simplifies the bioelectronic sensor design and opens unprecedented potential for bioelectronic sensor applications.

synthetic biology↗

bioRxiv2025

The growth benefits and toxicity of quinone synthesis are balanced by a dual regulatory mechanism and substrate limitations

Quinones play a central role in maintaining redox balance and conserving energy but can trigger oxidative stress at high levels. However, the mechanisms by which microbes regulate quinone levels remain poorly0020understood, hindering effective metabolic engineering to modulate microbes for quinone production. Here, we show that the synthesis of the menaquinone precursor DHNA in the lactic acid bacterium Lactococcus lactis is regulated by a combined genetic, enzymatic, and metabolic mechanism. Using synthetic biology approaches, we found that enzymes MenF and MenD both contribute to DHNA regulation, with MenD playing a more prominent role in controlling DHNA concentrations. A mathematical model elucidates a two-phase regulatory pattern resulting from the interplay of reversible flux and allosteric feedback inhibition, where either MenF or MenD can serve as the regulatory enzyme, depending on their relative expression ratio. Additionally, the overproduction of DHNA is constrained by substrate availability, ensuring a sufficient but not excessive DHNA level to benefit cell growth while mitigating cytotoxicity. Collectively, these mechanisms maintain a fine-tuned physiological quinone level and suggest that modulating substrate supplement and MenF-to-MenD ratio could be keys for engineering DHNA production

systems biology↗

bioRxiv2024

Extracellular Respiration is a Latent Energy Metabolism in Escherichia coli

Diverse microbial species utilize redox shuttles to exchange electrons with their environment through mediated extracellular electron transfer (EET). This process maintains redox homeostasis and supports anaerobic survival across diverse microbial communities. Although mediated EET has been extensively leveraged for bioelectrocatalysis and bioelectronics for decades, fundamental questions remain about how these redox shuttles are reduced within cells and their bioenergetic implications. This knowledge gap limits our understanding of the physiological roles of mediated EET in various microbes and hampers the development of efficient microbial electrochemical technologies. To address this, we developed a methodology integrating genome editing, electrochemistry, and systems biology to investigate the mechanism and bioenergetic implications of mediated EET in bacteria. Using this approach, we uncovered a mediated EET mechanism in Escherichia coli. In the absence of alternative electron sinks, the redox cycling of 2-hydroxy-1,4-naphthoquinone (HNQ) via a cytoplasmic nitroreductase enabled E. coli to respire and grow on an extracellular electrode. Genome-scale metabolic modeling suggested that HNQ- mediated EET offers a more energetically favorable route for supporting anaerobic growth than canonical fermentation. Transcriptome analysis revealed redox perturbations in response to HNQ and identified rapid metabolic adaptations that support growth. This work demonstrates that E. coli can grow independently of classical electron transport chains and fermentative pathways, unveiling a new type of anaerobic energy metabolism.

systems biology↗

bioRxiv2022

The differing roles of flavins and quinones in extracellular electron transfer in Lactiplantibacillus plantarum

Lactiplantibacillus plantarum is a lactic acid bacteria that is commonly found in the human gut and fermented food products. Despite its overwhelmingly fermentative metabolism, this microbe can perform extracellular electron transfer (EET) when provided with an exogenous quinone, 1,4-dihydroxy-2-naphthoic acid (DHNA) and riboflavin. However, the separate roles of DHNA and riboflavin in EET in L. plantarum has remained unclear. Here we seek to understand the role of quinones and flavins for EET by monitoring iron and anode reduction in the presence and absence of these small molecules. We found that either addition of DHNA or riboflavin can support robust iron reduction, indicating electron transfer to extracellular iron occurs through both flavin-dependent and DHNA-dependent routes. Using genetic mutants of L. plantarum, we found that flavin-dependent iron reduction requires Ndh2 and EetA, while DHNA-dependent iron reduction largely relies on Ndh2 and PplA. In contrast to iron reduction, DHNA-containing media supported more robust anode reduction than riboflavin-containing media, suggesting electron transfer to an anode proceeds most efficiently through the DHNA-dependent pathway. Furthermore, we found that flavin-dependent anode reduction requires EetA, Ndh2, and PplA, while DHNA-dependent anode reduction requires Ndh2 and PplA. Taken together, we identify multiple EET routes utilized by L. plantarum and show that the EET route depends on access to environmental biomolecules and on the extracellular electron acceptor. This work expands our molecular-level understanding of EET in Gram-positive microbes and provides additional opportunities to manipulate EET for biotechnology. ImportanceLactic acid bacteria are named because of their nearly exclusive fermentative metabolism. Thus, the recent observation of EET activity - typically associated with anaerobic respiration - in this class of organisms has forced researchers to rethink the rules governing microbial metabolic strategies. Our identification of multiple routes for EET in L. plantarum that depend on two separate redox active small molecules expands our understanding of how microbes metabolically adapt to different environments to gain an energetic edge and how these processes can be manipulated for biotechnological uses. Understanding the role of EET in lactic acid bacteria is of great importance due to the significance of lactic acid bacteria in agriculture, bioremediation, food production, and gut health. Furthermore, the maintenance of multiple EET routes speak to the importance of this process to function in a variety of environmental conditions.

microbiology↗

bioRxiv2022

Determinants of multiheme cytochrome extracellular electron transfer uncovered by systematic peptide insertion

The multiheme cytochrome MtrA enables microbial respiration by transferring electrons across the outer membrane to extracellular electron acceptors. While structural studies have identified residues that mediate MtrA binding to hemes and to other cytochromes that facilitate extracellular electron transfer (EET), the relative importance of these interactions for EET is not known. To better understand EET, we evaluated how insertion of an octapeptide across all MtrA backbone locations affects Shewanella oneidensis MR-1 respiration on Fe(III). EET efficiency was found to be inversely correlated with insertion proximity to the heme prosthetic groups. Mutants with decreased EET also arose from insertions in a subset of the regions that make residue-residue contacts with the porin MtrB, while all sites contacting the extracellular MtrC presented high peptide insertion tolerance. MtrA variants having peptide insertions within the CXXCH motifs that coordinate heme cofactors retained some ability to support respiration on Fe(III), although these variants presented significantly decreased EET. Furthermore, the fitness of cells expressing different MtrA variants under Fe(III)-respiring conditions correlated with anode reduction. The peptide-insertion profile, which represents the first comprehensive sequence-structure-function map for a multiheme cytochrome, implicates MtrA as a strategic protein engineering target for regulating EET.

synthetic biology↗

bioRxiv2021

Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD+ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1,000-fold in a mouse infection model. Consistent with NAD+ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX rescued this phenotype. These studies show that NAD+ regeneration, rather than oxidative phosphorylation, represents the primary role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.

microbiology↗

bioRxiv2021

Extracellular electron transfer increases fermentation in lactic acid bacteria via a hybrid metabolism

Energy conservation in microorganisms is classically categorized into respiration and fermentation, however recent work shows some species can use mixed or alternative bioenergetic strategies. We explored the utility of a flavin-based extracellular electron transport (FLEET) system for energy conservation within diverse lactic acid bacteria (LAB), microorganisms that mainly rely on fermentative metabolism and are important in food fermentations. The LAB Lactiplantibacillus plantarum uses extracellular electron transfer to increase its NAD+/NADH ratio, generate more ATP through substrate-level phosphorylation and accumulate biomass more rapidly. This novel, hybrid metabolism was dependent on a type-II NADH dehydrogenase (Ndh2) and conditionally required a flavin-binding extracellular lipoprotein (PplA) in the FLEET system to confer increased fermentation yield, metabolic flux, and environmental acidification in both laboratory media and food fermentation. The discovery of a single pathway that blends features of fermentation and respiration expands our knowledge of energy conservation metabolism and provides immediate biotechnology applications.

microbiology↗

bioRxiv2020

A hybrid cyt c maturation system enhances the bioelectrical performance of engineered Escherichia coli by improving the rate-limiting step

Bioelectronic devices can use electron flux to enable communication between biotic components and abiotic electrodes. We have modified Escherichia coli to electrically interact with electrodes by expressing the cytochrome c from Shewanella oneidensis MR-1. However, we observe inefficient electrical performance, which we hypothesize is due to the limited compatibility of the E. coli cytochrome c maturation (Ccm) systems with MR-1 cytochrome c. Here we test whether the bioelectronic performance of E. coli can be improved by constructing hybrid Ccm systems containing protein domains from both E. coli and S. oneidensis MR-1. The hybrid CcmH increased cytochrome c expression by increasing the abundance of CymA 60%, while only slightly changing the abundance of the other cytochromes c. Electrochemical measurements showed that the overall current from the hybrid ccm strain increased 121% relative to the wildtype ccm strain, with an electron flux per cell of 12.3 {+/-} 0.3 fA{middle dot}cell-1. Additionally, the hybrid ccm strain doubled its electrical response with the addition of exogenous flavin, and quantitative analysis of this demonstrates CymA is the rate-limiting step in this electron conduit. These results demonstrate that this hybrid Ccm system can enhance the bioelectrical performance of the cyt c expressing E. coli, allowing the construction of more efficient bioelectronic devices.

bioengineering↗

bioRxiv2020

Precise electronic control of redox reactions inside Escherichia coli using a genetic module

Microorganisms regulate the redox state of different biomolecules to precisely control biological processes. These processes can be modulated by electrochemically coupling intracellular biomolecules to an external electrode, but current approaches afford only limited control and specificity. Here we describe specific electrochemical control of the reduction of intracellular biomolecules in Escherichia coli through introduction of a heterologous electron transfer pathway. E. coli expressing mtrCAB from Shewanella oneidensis MR-1 consumed electrons directly from a cathode when fumarate or nitrate, both intracellular electron acceptors, were present. The fumarate-triggered current consumption occurred only when fumarate reductase was present, indicating all the electrons passed through this enzyme. Moreover, MtrCAB-expressing E. coli used current to stoichiometrically produce ammonia. Thus, our work introduces a modular genetic tool to reduce a specific intracellular redox molecule with an electrode, opening the possibility of electronically controlling biological processes such as biosynthesis and growth in any microorganism.

synthetic biology↗