Biology subjects

Hayes, J. A.

Publications and source records attributed to Hayes, J. A..

8 recordsLinked to original sources

bioRxiv2026

13C-Metabolic Flux Analysis of Substrate Co-Metabolism in the Methylotrophic Acetogen Eubacterium limosum.

Acetogens are promising microbial catalysts for the sustainable production of fuels and chemicals, metabolizing a range of renewable single-carbon (C1) compounds with high energetic efficiency. The major challenge is their energy-limited anaerobic lifestyle, which results in slow growth and a limited product spectrum on individual C1 substrates. We previously showed in the acetogen Eubacterium limosum that co-metabolism of glucose-methanol or CO-methanol mixtures results in substantial improvements in growth and acetate productivity. To investigate the underlying mechanism(s), here we conducted 13C metabolic flux analysis (13C-MFA) of E. limosum grown on a glucose-methanol mixture, as well as in single-substrate controls. The results suggest that the main benefit of glucose cofeeding is not provision of additional ATP, but rather a reduction of flux through the ferredoxin-dependent and kinetically challenging reactions of the Wood-Ljungdahl pathway. Subsequent RNAseq analysis showed no significant differences in the expression level of genes involved in methanol metabolism between cofeeding and methanol-only controls, suggesting this reduction of flux is controlled post-transcriptionally. Most surprisingly, we found that under all growth conditions, flux through pyruvate:ferredoxin oxidoreductase (PFOR) was in the oxidative direction, with cysteine (added as a reducing agent) providing all required gluconeogenic carbon flux under CO2-fixing conditions. This work provides critical insights into the metabolic network of an emerging model acetogen that will facilitate future metabolic engineering efforts to channel flux to value-added products, and highlights the under-appreciated role of cysteine as a carbon source in acetogenic fermentations.

systems biology↗

bioRxiv2026

Metabolic engineering of Escherichia coli to modulate hydrogen sulfide levels in the mammalian gut

Hydrogen sulfide (H2S) is a microbiota-derived metabolite in the gastrointestinal tract implicated in a number of diseases. Its volatility and reactivity make experimentally controlling H2S concentration in vivo difficult, limiting our ability to interrogate its dose-dependent effects on host physiology. Engineered bacteria present a compelling solution, yet most probiotic metabolic engineering approaches have focused on in vitro optimization, failing to account for the complex intestinal environment. Here, we engineered Escherichia coli strains to produce or consume H2S in specific intestinal regions by incorporating knowledge of the local metabolic environment and resident microbial activities into the design process. Analysis of human-derived ex vivo cultures revealed that glutathione (GSH) is inefficiently converted to H2S, suggesting GSH as a relatively stable substrate for engineered sulfide production. We thus engineered a GSH-dependent H2S producer, which increased levels 21-fold ex vivo. To target the nutrient-rich, microbially sparse environment of the small intestine, we optimized a H2S producer that uses L-cysteine as a sulfur source, demonstrating a 7-fold increase in H2S levels in mice. Finally, to develop strains capable of sequestering H2S, we leveraged the availability of fumarate and nitrate as electron acceptors in the large intestine by engineering a strain expressing sulfide:quinone oxidoreductase (Sqr). This enables oxidation of H2S to intracellular polysulfides and achieves higher consumption rates than alternative sequestration strategies reliant on resource-intensive GSH production. Together, this work developed engineered microbes as precision tools to modulate H2S levels and showcases a generalizable framework for region-targeted design of engineered probiotics.

synthetic biology↗

bioRxiv2024

Modeling Insights into Potential Mechanisms of Opioid-Induced Respiratory Depression within Medullary and Pontine Networks

The opioid epidemic is a pervasive health issue and continues to have a drastic impact on the healthcare system and United States. This is primarily because opioids cause respiratory suppression and the leading cause of death in opioid overdose is respiratory failure (i.e., opioid-induced respiratory depression, OIRD). Opioid administration can affect the frequency and magnitude of inspiratory motor drive by activating {micro}-opioid receptors that are located throughout the respiratory control network in the brainstem. This can significantly affect ventilation and blunt CO2 responsiveness, but the precise neural mechanisms that suppress breathing are not fully understood. Previous research has suggested that opioids affect medullary and pontine inspiratory neuron activity by disrupting upstream elements within this circuit. Inspiratory neurons within this network exhibit synchrony consistent with shared excitation from other neuron populations and recurrent mechanisms. One possible target for opioid suppression of inspiratory drive are excitatory synapses. Reduced excitability of these synaptic elements may result in disfacilitation and reduced synchrony among inspiratory neurons. Downstream effects of disfacilitation may result in abnormal output from phrenic motoneurons resulting in distressed breathing. We tested the plausibility of this hypothesis with a computational model of the respiratory network by targeting the synaptic excitability in fictive medullary and pontine populations. The synaptic conductances were systematically decreased while monitoring the overall respiratory motor pattern and aggregate firing rates of subsets of cell populations. Simulations suggest that highly selective, rather than generalized, actions of opioids on synapses within the inspiratory network may account for different observed breathing mechanics.

physiology↗

bioRxiv2023

Evidence for peripheral and central actions of codeine to dysregulate deglutition in the anesthetized cat

Systemic administration of opioids has been associated with aspiration and swallow dysfunction in humans. We speculated that systemic administration of codeine would induce dysfunctional swallowing and that this effect would have a peripheral component. Experiments were conducted in spontaneously breathing, anesthetized cats. The animals were tracheotomized and electromyogram electrodes were placed in upper airway and chest wall respiratory muscles for recording swallow related motor activity. The animals were allocated into three groups: vagal intact (VI), cervical vagotomy (CVx), and supra-nodose ganglion vagotomy (SNGx). A dose response to intravenous codeine was performed in each animal. Swallowing was elicited by injection of 3 ml of water into the oropharynx. The number of swallows after vehicle was significantly higher in the VI group than in SNGx. Codeine had no significant effect on the number of swallows induced by water in any of the groups. However, the magnitudes of water swallow-related EMGs of the thyropharyngeus muscle were significantly increased in the VI and CVx groups by 2-4 fold in a dose-related manner. In the CVx group, the geniohyoid muscle EMG during water swallows was significantly increased. There was a significant codeine dose-related increase in spontaneous swallowing in each group. The spontaneous swallow number at the 10 mg/kg dose of codeine was significantly larger in the CVx than that in the SNGx groups. During water-evoked swallows, intravenous codeine increased upper airway motor drive in a dose-related manner, consistent with dysregulation. The appearance of spontaneous swallowing in response to codeine in all groups supports a central action of this drug on the swallow pattern generator and also is consistent with dysregulation. At the highest dose of codeine, the reduced spontaneous swallow number in the SNGx group relative to CVx is consistent with a peripheral excitatory action of codeine either on pharyngeal/laryngeal receptors or in the nodose ganglion itself.

physiology↗

bioRxiv2023

Engineered bacteria titrate hydrogen sulfide and induce concentration-dependent effects on host in a gut microphysiological system

Hydrogen sulfide (H2S) is a gaseous microbial metabolite whose role in gut diseases is debated, largely due to the difficulty in controlling its concentration and the use of non-representative model systems in previous work. Here, we engineered E. coli to titrate H2S controllably across the physiological range in a gut microphysiological system (chip) supportive of the co-culture of microbes and host cells. The chip was designed to maintain H2S gas tension and enable visualization of co-culture in real-time with confocal microscopy. Engineered strains colonized the chip and were metabolically active for two days, during which they produced H2S across a sixteen-fold range and induced changes in host gene expression and metabolism in an H2S concentration-dependent manner. These results validate a novel platform for studying the mechanisms underlying microbe-host interactions, by enabling experiments that are infeasible with current animal and in vitro models.

bioengineering↗

bioRxiv2021

Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader

Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the S. cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale crab-claw expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFCs switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.

biochemistry↗

bioRxiv2019

A thermophilic phage uses a small terminase protein with a fixed helix-turn-helix geometry

Tailed bacteriophage use a DNA packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component acts as a molecular matchmaker by recognizing the viral genome as well as the main motor component, the large terminase (TerL). How TerS binds DNA and the TerL protein remains unclear. Here, we identify the TerS protein of the thermophilic bacteriophage P74-26. TerSP76-26 oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. Our cryo-EM structure shows that TerSP76-26 forms a ring with a wide central pore and radially arrayed helix-turn-helix (HTH) domains. These HTH domains, which are thought to bind DNA by wrapping the helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA binding domain imposes strong constraints on how TerSP76-26 can bind DNA. Finally, the TerSP76-26 structure lacks the conserved C-terminal {beta}-barrel domain used by other TerS proteins for binding TerL, suggesting that a well-ordered C-terminal {beta}-barrel domain is not necessary for TerS to carry out its function as a matchmaker.

biochemistry↗

bioRxiv2016

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the ATPase that powers DNA translocation and an endonuclease that cleaves the concatemeric genome both at initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage is still mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nucleolysis. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of nucleolysis suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid shell during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the procapsid upon completion of packaging unlocks the nuclease domains to cleave DNA.

biochemistry↗