Biology subjects

Kennedy, N. W.

Publications and source records attributed to Kennedy, N. W..

15 recordsLinked to original sources

bioRxiv2026

Distribution of luxS and production of autoinducer-2 among gut Bacteroidales

One of the best studied quorum sensing (QS) molecules, autoinducer-2 (AI-2), regulates processes in numerous bacteria. LuxS is an enzyme of the activated methyl cycle which, along with MtnN, converts S-adenosyl-homocysteine to homocysteine, releasing DPD (4,5-dihydroxy-2,3-pentanedione) which is spontaneously converted to AI-2. Many bacteria do not encode MtnN-LuxS and instead encode SahH, which directly converts S-adenosyl-homocysteine to homocysteine without AI-2 production. The genomes of some gut Bacteroidales were shown to contain luxS, however, these reports, as well as reports of the production of AI-2 by gut Bactereoidales have been inconsistent. We performed a comprehensive analysis of the distribution of luxS and sahH in Bacteroidota with an in-depth exploration of gut Bacteroidales. The data suggest that the ancestral Bacteroidota contained sahH, with numerous independent replacements with mtnN-luxS during diversification. In Bacteroidaceae, Parabacteroides, and many Prevotellaceae, mtnN-luxS or sahH are present in the same genetic region, adjacent to yfhO. Using Bacteroides fragilis, which contains sahH, and Bacteroides uniformis and Phocaeicola vulgatus, whose genomes contain mtnN-luxS, we show that luxS-containing strains produce AI-2. Transcriptomic analyses and gnotobiotic mouse experiments using wild-type strains and mtnN-luxS and sahH genetic swaps showed that Bacteroidaceae species do not respond to AI-2 under the conditions tested. However, analyses of 15 human gut metagenomic datasets show that mtnN-luxS containing Bacteroidales species average between 5.1 - 33% of the total bacteria in these various human gut microbiome datasets, and therefore, likely produce substantial amounts of AI-2 sensed by responsive gut symbionts and pathogens. ImportanceHere, we show that gut Bacteroidales possess one of two pathways to complete the activated methyl cycle, one that produces the QS molecule AI-2. We clarify conflicting data regarding AI-2 production in gut Bacteroidales and show that luxS is present in many gut Bacteroidales species, but lacking in species such as Bacteroides thetaiotaomicron and Bacteroides fragilis. We find that prevalent and abundant human gut Bacteroidales species including Bacteroides uniformis and Phocaeicola vulgatus produce substantial amounts of AI-2. While our data do not show that Bacteroidales sense or respond to AI-2 under the conditions tested, analyses of human gut metagenomic data reveal that AI-2 producing Bacteroidales comprise a large proportion of the gut bacteria of both industrialized and non-industrialized human populations.

microbiology↗

bioRxiv2026

Glycerol alone effects 1,3-propanediol production via the aerobic propanediol utilization pathway in Salmonella enterica

Crude glycerol is an underutilized waste stream. Viable routes for converting it to 1,3-propanediol (1,3-PDO) can conserve important resources and add value to its supply chain. Biological methods are appealing because they can circumvent expensive preprocessing steps while operating under mild conditions. Here, we show that the propanediol utilization pathway of Salmonella enterica serovar Typhimurium LT2 can be used to convert glycerol, including unprocessed crude glycerol, into 1,3-PDO under aerobic conditions in minimal media. Additionally, we demonstrate that high concentrations of expensive cofactors are not necessary to achieve optimal production titers. This study lays the groundwork for continual iteration on this pathway for bioprocess development. Key pointsO_LIS. enterica can produce 1,3-propanediol from crude glycerol alone C_LIO_LIGlycerol-to-1,3-propanediol conversion is dependent on expression of the propanediol utilization (Pdu) pathway C_LIO_LISub-saturating concentrations of exogenous vitamin B12 can boost cell growth and 1,3-propanediol yield C_LI

microbiology↗

bioRxiv2025

Determinants of Protein-Level Parameters Governing MS2 VLP Reassembly

MS2 virus-like particles (VLPs) are widely used as protein nanocages for cargo encapsulation, yet in vitro disassembly-reassembly protocols remain poorly standardized, and reassembly yields are reported inconsistently. As a result, the same experiments reported in literature produce widely divergent yields, limiting reproducibility and cross-study comparability. Here, we introduce a cargo-specific, quantitative framework for standardized MS2 VLP reassembly yield determination. We evaluate commonly used disassembly and post-disassembly processing methods and identify practical trade-offs between protein recovery, accessibility, and reproducibility. Reassembly yield is quantified using size exclusion chromatography calibrated against purified VLP standards, enabling robust, cargo-specific yield measurement. Using this framework, we apply a full factorial design of experiments to quantify the individual and combined effects of coat protein concentration, ionic strength, buffer pH, and molecular crowding on reassembly yield. The resulting statistical model explains more than 99% of the explainable variance and its linear fit to the experimental data indicates that optimal reassembly conditions extend beyond those tested to date. Protein concentration and ionic strength dominate reassembly yield, whereas pH and osmolyte concentration contribute more modestly within the tested ranges. Finally, we propose practical guidelines for standardized MS2 VLP disassembly, reassembly, and yield reporting, defining a transferable operating envelope for MS2 VLP reconstruction. While demonstrated here using a single nucleic acid cargo (tr-DNA), the framework is readily extensible to alternative cargos and coat protein variants.

biophysics↗

bioRxiv2025

A high throughput flow cytometry assay for quantifying type 3 secretion system assembly in Salmonella

Recombinant protein production in bacteria is limited by costly cell lysis and multi-step purification. To bypass these limitations, leveraging native bacterial secretion systems to secrete proteins offers a promising alternative. The type 3 secretion system (T3SS) of Salmonella enterica serovar Typhimurium can secrete proteins at hundreds of milligrams per liter. While promising, higher titers from this and other natural systems are needed to be relevant commercially. A major engineering target to enhance protein secretion is the T3SS secretion apparatus. However, efforts to quantify its assembly are hindered by bottlenecks in scalability and complexity. Assembly involves [~]20 structural proteins forming with precise stoichiometry under dynamic regulation. Membrane- and periplasmic-embedded components are difficult to probe without costly, time-intensive methods. To fully realize the T3SS as a tool for scalable protein production, new tools are needed for rapid and accurate characterization of the assembled apparatus. Here, we establish a high throughput flow cytometry method in S. Typhimurium by quantifying the abundance of the needle-bound tip protein SipD as a proxy for T3SS apparatus assembly. We then adapted a super resolution microscopy method, known as Structured Illumination Microscopy (SR-SIM), to visualize the presence of SipD and validate the flow cytometry results. Applying this approach, we revealed how overexpression of key T3SS regulators hilA and hilD impact assembly, expanded the assay with a secretion-compatible fluorescent reporter to link assembly with secretion, and uncovered how a PrgI variant impacts apparatus architecture. Together, these tools enable rapid insights into T3SS assembly and advance heterologous secretion platform development. ImportanceProducing proteins in bacteria often requires expensive and complex steps. This study focuses on S. Typhimurium, a bacterium that naturally secretes proteins using a specialized system. We developed a fast and scalable method to measure assembly of the secretion machinery, ultimately accelerating the process of optimizing this system for bacterial protein production. Further, this method provides new mechanistic insights into how the changes to the system impact the ability for the secretion machinery to form. We expect the platform could be adopted to other systems displayed on the bacterial cell surface, including T3SS homologs as well as a wide variety of multi-component membrane transporters.

synthetic biology↗

bioRxiv2025

Self-assembling protein materials with genetically programmable morphology and size

Materials are challenging to synthetically program down to the atom level. Nature, however, excels at creating hierarchical materials from nanoscale building blocks, a feat that remains a major challenge in synthetic systems. A deeper understanding of the molecular rules governing self-assembly would unlock the potential for designing genetically programmable materials with atomic precision. Hexameric bacterial microcompartment (BMC-H) proteins offer a powerful model system for exploring this question. These sequence-defined proteins naturally assemble into complex architectures and can be expressed biologically, making them ideal candidates for studying how minor sequence variations influence supramolecular structure. In this work, we leverage cell-free protein synthesis (CFPS) alongside immunostaining and super-resolution microscopy to investigate the self-assembly behavior of two BMC-H proteins, PduA and PduJ. We find that both proteins form micro-to millimeter scale structures when expressed in vitro. Further, we demonstrate how single point mutation changes lead PduA and PduJ to form significantly different supramolecular structures when produced using CFPS. These studies support the future exploration of self-assembling proteins as programmable scaffolds in broad materials applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/666636v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@3376faorg.highwire.dtl.DTLVardef@c81730org.highwire.dtl.DTLVardef@6a6a5borg.highwire.dtl.DTLVardef@6cb4ff_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Synthetic Rewiring of Virus-Like Particles via Circular Permutation Enables Modular Peptide Display and Protein Encapsulation

Virus-like particles (VLPs) are self-assembling nanoparticles derived from viruses with potential as scaffolds for myriad applications. They are also excellent testbeds for engineering protein superstructures. Engineers often employ techniques such as amino acid substitutions and insertions/deletions. Yet evolution also utilizes circular permutation, a powerful natural strategy that has not been fully explored in engineering self-assembling protein nanoparticles. Here, we demonstrate this technique using the MS2 VLP as a model self-assembling, proteinaceous nanoparticle. We constructed, for the first time, a comprehensive circular permutation library of the fused MS2 coat protein dimer construct. The strategy revealed new terminal locations, validated via cryo-electron microscopy, that enabled C-terminal peptide tagging and led to a stable protein encapsulation strategy via covalent bonding - a feature the native coat protein does not permit. Our systematic study demonstrates the power of circular permutation for engineering new features as well as quantitatively and systematically exploring VLP structural determinants.

synthetic biology↗

bioRxiv2024

Signal sequences target enzymes and structural proteins to bacterial microcompartments and are critical for microcompartment formation

Spatial organization of pathway enzymes has emerged as a promising tool to address several challenges in metabolic engineering, such as flux imbalances and off-target product formation. Bacterial microcompartments (MCPs) are a spatial organization strategy used natively by many bacteria to encapsulate metabolic pathways that produce toxic, volatile intermediates. Several recent studies have focused on engineering MCPs to encapsulate heterologous pathways of interest, but how this engineering affects MCP assembly and function is poorly understood. In this study, we investigated the role of signal sequences, short domains that target proteins to the MCP core, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterized two novel Pdu signal sequences on the structural proteins PduM and PduB, which constitutes the first report of metabolosome signal sequences on structural proteins rather than enzymes. We then explored the role of enzymatic and structural Pdu signal sequences on MCP assembly by deleting their encoding sequences from the genome alone and in combination. Deleting enzymatic signal sequences decreased MCP formation, but this defect could be recovered in some cases by overexpressing genes encoding the knocked-out signal sequence fused to a heterologous protein. By contrast, deleting structural signal sequences caused similar defects to knocking out the genes encoding the full length PduM and PduB proteins. Our results contribute to a growing understanding of how MCPs form and function in bacteria and provide strategies to mitigate assembly disruption when encapsulating heterologous pathways in MCPs.

synthetic biology↗

bioRxiv2024

Characterization and Engineering of the Type 3 Secretion System Needle Monomer from Salmonella Through the Construction and Screening of a Comprehensive Mutagenesis Library

Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here we investigated this link with the PrgI protein, the monomer of the secretory channel of the Type 3 Secretion System (T3SS) of Salmonella enterica. Despite detailed knowledge of the PrgI needles assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape (SFL) to characterize the PrgI needles mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SSs assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes. ImportanceProtein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system from Salmonella enterica can impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system.

bioengineering↗

bioRxiv2023

The variable domain from the mitochondrial fission mechanoenzyme Drp1 promotes liquid-liquid phase separation

Dynamins are an essential superfamily of mechanoenzymes that remodel membranes and often contain a "variable domain" (VD) important for regulation. For the mitochondrial fission dynamin, Drp1, a regulatory role for the VD is demonstrated by mutations that can elongate, or fragment, mitochondria. How the VD encodes inhibitory and stimulatory activity is unclear. Here, isolated VD is shown to be intrinsically disordered (ID) yet undergoes a cooperative transition in the stabilizing osmolyte TMAO. However, the TMAO stabilized state is not folded and surprisingly appears as a condensed state. Other co-solutes including known molecular crowder Ficoll PM 70, also induce a condensed state. Fluorescence recovery after photobleaching experiments reveal this state to be liquid-like indicating the VD undergoes a liquid-liquid phase separation under crowding conditions. These crowding conditions also enhance binding to cardiolipin, a mitochondrial lipid, raising the possibility that phase separation may enable rapid tuning of Drp1 assembly necessary for fission.

biophysics↗

bioRxiv2021

Vertex protein PduN tunes encapsulated pathway performance by dictating bacterial metabolosome morphology

Engineering subcellular organization in microbes shows great promise in addressing bottlenecks in metabolic engineering efforts; however, rules guiding selection of an organization strategy or platform are lacking. Here, we study compartment morphology as a factor in mediating encapsulated pathway performance. Using the 1,2-propanediol utilization microcompartment (Pdu MCP) system from Salmonella enterica serovar Typhimurium LT2, we find that we can shift the morphology of this protein nanoreactor from polyhedral to tubular by removing vertex protein PduN. Analysis of the metabolic function between these Pdu microtubes (MTs) shows that they provide a diffusional barrier capable of shielding the cytosol from a toxic pathway intermediate, similar to native MCPs. However, kinetic modeling suggests that the different surface area to volume ratios of MCP and MT structures alters encapsulated pathway performance. Finally, we report a microscopy-based assay that permits rapid assessment of Pdu MT formation to enable future engineering efforts on these structures.

bioengineering↗

bioRxiv2021

Linking the Salmonella enterica 1,2-propanediol utilization bacterial microcompartment shell to the enzymatic core via the shell protein PduB

Bacterial microcompartments (MCPs) are protein-based organelles that house the enzymatic machinery for metabolism of niche carbon sources, allowing enteric pathogens to outcompete native microbiota during host colonization. While much progress has been made toward understanding MCP biogenesis, questions still remain regarding the mechanism by which core MCP enzymes are enveloped within the MCP protein shell. Here we explore the hypothesis that the shell protein PduB is responsible for linking the shell of the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica serovar Typhimurium LT2 to its enzymatic core. Using fluorescent reporters, we demonstrate that all members of the Pdu enzymatic core are encapsulated in Pdu MCPs. We also demonstrate that PduB is the sole protein responsible for linking the entire Pdu enzyme core to the MCP shell. Using MCP purifications, transmission electron microscopy, and fluorescence microscopy we find that shell assembly can be decoupled from the enzymatic core, as apparently empty MCPs are formed in Salmonella strains lacking PduB. Mutagenesis studies also reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and systems-level pathway modeling reveal that unencapsulated pathway performance is strongly impacted by enzyme concentration, highlighting the importance of minimizing polar effects when conducting these functional assays. Together, these results provide insight into the mechanism of enzyme encapsulation within Pdu MCPs and demonstrate that the process of enzyme encapsulation and shell assembly are separate processes in this system, a finding that will aid future efforts to understand MCP biogenesis. ImportanceMCPs are unique, genetically encoded organelles used by many bacteria to survive in resource-limited environments. There is significant interest in understanding the biogenesis and function of these organelles, both as potential antibiotic targets in enteric pathogens and also as useful tools for overcoming metabolic engineering bottlenecks. However, the mechanism by which these organelles are formed natively is still not completely understood. Here we provide evidence of a potential mechanism inS. enterica by which a single protein, PduB, links the MCP shell and metabolic core. This finding is critical for those seeking to disrupt MCPs during pathogenic infections or for those seeking to harness MCPs as nanobioreactors in industrial settings.

synthetic biology↗

bioRxiv2021

Density-based binning of gene clusters to infer function or evolutionary history using GeneGrouper

MotivationIdentifying gene clusters of interest in phylogenetically proximate and distant taxa can help to infer phenotypes of interest. Conserved gene clusters may differ by only a few genes, which can be biologically meaningful, such as the formation of pseudogenes or insertions interrupting regulation. These qualities may allow for unsupervised clustering of similar gene clusters into bins that provide a population-level understanding of the genetic variation in similar gene clusters. ResultsWe developed GeneGrouper, a command-line tool that uses a density-based clustering method to group gene clusters into bins. GeneGrouper demonstrated high recall and precision in benchmarks for the detection of the 23-gene Salmonella enterica LT2 Pdu gene cluster and four-gene Pseudomonas aeruginosa PAO1 Mex gene cluster in 435 genomes containing mixed taxa. In a subsequent application investigating the diversity and impact of gene complete and incomplete LT2 Pdu gene clusters in 1130 S. enterica genomes, GeneGrouper identified a novel, frequently occurring pduN pseudogene. When replicated in vivo, disruption of pduN with a frameshift mutation negatively impacted microcompartment formation. We next demonstrated the versatility of GeneGrouper by clustering both distant homologous gene clusters and variable gene clusters found in integrative and conjugative elements. AvailabilityGeneGrouper software and code are publicly available at https://github.com/agmcfarland/GeneGrouper.

bioinformatics↗

bioRxiv2021

Computational and experimental approaches to controlling bacterial microcompartment assembly

Bacterial microcompartments compartmentalize the enzymes that aid chemical and energy production in many bacterial species. These protein organelles are found in various bacterial phyla and are postulated to help many of these organisms survive in hostile environments such as the gut of their hosts. Metabolic engineers are interested in repurposing these organelles for non-native functions. Here, we use computational, theoretical and experimental approaches to determine mechanisms that effectively control microcompartment self-assembly. As a model system, we find via multiscale modeling and mutagenesis studies, the interactions responsible for the binding of hexamer-forming proteins propanediol utilization bacterial microcompartments from Salmonella and establish conditions that form various morphologies. We determine how the changes in the microcompartment hexamer protein preferred angles and interaction strengths can modify the assembled morphologies including the naturally occurring polyhedral microcompartment shape, as well as other extended shapes or quasi-closed shapes. We demonstrate experimentally that such altered strengths and angles are achieved via amino acid mutations. A thermodynamic model that agrees with the coarse-grained simulations provides guidelines to design microcompartments. These findings yield insight in controlled protein assembly and provide principles for assembling microcompartments for biochemical or energy applications as nanoreactors.

biophysics↗

bioRxiv2020

Self-assembling shell proteins PduA and PduJ have essential and redundant roles in bacterial microcompartment assembly

Protein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts.

microbiology↗

bioRxiv2019

Apparent size and morphology of bacterial microcompartments varies with technique

Bacterial microcompartments (MCPs) are protein-based organelles which encapsulate metabolic pathways. Metabolic engineers have recently sought to repurpose MCPs to encapsulate heterologous pathways to increase flux through pathways of interest. As MCP engineering becomes more common, standardized methods for analyzing changes to MCPs and interpreting results across studies will become increasingly important. In this study, we demonstrate that different imaging techniques yield variations in the apparent size of purified MCPs from Salmonella enterica serovar Typhimurium LT2, likely due to variations in sample preparation methods. We provide guidelines for preparing samples for MCP imaging and outline expected variations in apparent size and morphology between methods. With this report we aim to establish an aid for comparing results across studies.

synthetic biology↗