Biology subjects

Grinter, R.

Publications and source records attributed to Grinter, R..

At least 19 recordsLinked to original sources

bioRxiv2026

A diverse family of protein antibiotics inhibits the BAM complex by β-signal mimicry

Lectin-like protein antibiotics (Llps) kill Pseudomonas by inhibiting BamA, the essential core of the beta-barrel assembly machinery (BAM). Llps are composed of one or two beta;-lectin domains and a C-terminal peptide, with characterisation to date centred on the two-domain L-type pyocins of P. aeruginosa, which bind BamA extracellular loop 6 and inhibit by delivering the C-terminal peptide to BamA beta;-strand 1. Whether this mechanism is general, and how Llps contend with BamA sequence differences across species, remained unknown. We screened 238 Pseudomonas Llps against 101 genome-sequenced isolates, showing susceptibility across the family is set by BamA extracellular loop 6. Loop 6 length varies, and Llps fall into three targeting modes: short-loop and long-loop specialists and broad-range dual targeters, which have arisen repeatedly. We determine the 2.66 angstrom cryo-electron microscopy structure of a two-domain LlpA bound to BAM, showing recognition has been relocated relative to the L-type pyocins, with loop 6 bound by a single face of the N-terminal beta-lectin domain rather than the inter-domain cleft. This explains how single-domain LlpBs retain BamA targeting and why the second lectin domain has been lost at least twice. We show that the BamA inhibition mechanism is shared across Llps, with a hypervariable C-terminal peptide binding BamA beta-strand 1, inhibiting the BAM insertase by mimicking the beta-signal of BamA substrates. Together, these data provide an atlas of Llp diversity and the mechanistic principles for engineering it, identifying Llps as a tuneable, multi-interface scaffold for BAM-directed precision antibacterials against pathogenic Pseudomonas, including economically important plant pathogens.

biochemistry↗

bioRxiv2026

Diverse and novel lanthanide-binding PQQ-dependent enzymes

First described pyrroloquinoline quinone-dependent (PQQ) eight-bladed {beta}-propeller proteins are calcium (Ca)-dependent, but many homologous bacterial enzymes are lanthanide (Ln)-dependent. Discovery of Ca-dependent six-bladed {beta}-propeller PQQ-dependent dehydrogenases motivated the search for Ln-dependent six-bladed {beta}-propeller PQQ-dependent enzymes in bacteria. Using in silico structural prediction of sequences from weathered rock, we identified [~]22,000 PQQ six-bladed {beta}-propeller proteins in bacteria from 77 phyla, of which 63% of sequences have the active site residues needed to bind Ln. PQQ and La binding was biochemically confirmed for enzymes from uncultivated Chloroflexi and Acidobacteria. In structural models, Ln-binding periplasmic proteins interact with TonB-dependent transporters that may enable Ln uptake. Most genomes also encode predicted Ln-and PQQ-dependent eight-bladed dehydrogenases that clade with diverse alcohol and sugar dehydrogenases. Thus, Ln-dependent six and eight-bladed PQQ-dependent {beta}-propeller proteins are implicated in diverse carbon substrate metabolisms in weathering rock and soil. We predict that many PQQ-dependent microbial enzymes are lanthanide dependent.

microbiology↗

bioRxiv2026

Quinone-transporting filaments extend the respiratory chain of Gram positive bacteria

Cellular respiration depends on transferring electrons to hydrophobic quinones in membrane bilayers, constraining capacity to available surface area. Expanding this capacity is thought to have driven cellular complexity and eukaryogenesis, with Gram-negative bacteria evolving internal invaginations and eukaryotes using membrane-bound organelles. Whether Gram-positive bacteria, which lack such membranes, evolved alternatives was unknown. Here, we show that Bacillus subtilis forms a quinone-transporting pseudomembrane composed of filaments of the NADH dehydrogenase Ndh and the quinone-transporting protein Ncp. Cryo-EM, lipidomics, and molecular dynamics reveal that Ndh and Ncp co-assemble with phospholipids into a complex containing a solvent-excluded hydrophobic lumen that sequesters quinones. These complexes further assemble into filaments, linking chambers into a continuous conduit that amplifies quinone reduction while occupying minimal membrane space. Phylogenetic analysis suggests this recent innovation is widespread in Bacillota. Quinone-transporting filaments thus reveal a third strategy for overcoming surface-area limits and provide principles for engineering synthetic energy systems.

biochemistry↗

bioRxiv2026

A complete RXFP1-relaxin interaction model unlocks the design of potent mini-protein modulators

Relaxin family peptide receptor 1 (RXFP1) is a multi-domain GPCR with compelling therapeutic potential, yet uncertainty surrounding the mechanism of its activation by the hormone H2 relaxin has hindered the development of selective modulators. Here, we combine deep learning based structural modelling with de novo protein design to overcome this barrier. We generate a high-confidence structural model of the RXFP1-relaxin complex that is strongly supported by existing biochemical and functional evidence. This model reveals that relaxin engagement stabilises the RXFP1 extracellular linker, thereby triggering receptor activation. Guided by this model, we design mini-protein modulators that either block linker stabilisation or enforce it and induce an active receptor geometry. These molecules act as potent, selective RXFP1 antagonists or agonists, achieving low-nanomolar activity in both engineered and endogenously expressing cell lines despite adopting folds unrelated to relaxin. Together, these findings define the mechanistic basis of RXFP1 signalling, establish the first de novo agonists and antagonists of this receptor, and demonstrate how AI-enabled modelling and design can target structurally complex GPCRs previously inaccessible to structure-guided drug discovery.

biochemistry↗

bioRxiv2026

Cryo-EM structure of human LRRC15 reveals the basis of therapeutic antibody recognition

Leucine-rich repeat containing 15 (LRRC15) is a structural uncharacterised membrane protein, which is highly upregulated in cancer-associated fibroblasts and enriched in desmoplastic tumours including pancreatic, breast and head-and-neck cancers, where it is associated with therapy resistance. The LRRC15-targeting antibody-drug conjugate samrotamab vedotin has advanced into Phase I clinical evaluation, yet mechanistic understanding of how samrotamab engages its target has remained elusive in the absence of structural data. Here, using hydrogen-deuterium exchange mass spectrometry and cryo-electron microscopy, we define the samrotamab epitope on LRRC15 and determine the structure of the LRRC15-samrotamab complex to 2.6 [A] resolution - the first high-resolution structure of this receptor. Samrotamab engages a membrane-proximal epitope within the C-terminal leucine-rich repeat region, leaving the canonical concave surface fully exposed for potential interactions. The lateral binding mode of samrotamab and the open arc geometry of LRRC15 provide a structural rationale for why antibody occupancy may leave signalling-competent surfaces intact. Guided by these insights, we computationally designed minibinders targeting the concave surface, identifying multiple binders with nanomolar affinity. Together, our work establishes the first structural framework for LRRC15, defines the molecular basis of therapeutic antibody recognition, and identifies the concave surface as a promising target for next-generation LRRC15-directed therapeutics.

biochemistry↗

bioRxiv2025

Atmospheric hydrogen consumption is regulated by catabolite repression in mycobacteria

Consumption of atmospheric hydrogen (H2) enables diverse aerobic microorganisms to grow and persist in resource-deprived environments. In the aerobic saprophyte Mycobacterium smegmatis, hydrogen oxidation is catalyzed by two differentially expressed, high-affinity, oxygen-insensitive uptake hydrogenases, Huc and Hhy. Huc enables mixotrophic growth and facilitates the transition from growth to dormancy. Although the huc operon is known to be upregulated in response to organic carbon deprivation, the specific signals and regulators modulating its expression remain unresolved. Here, we show that GylR, a glycerol-3-phosphate-sensing regulator of glycerol metabolism, plays a role in repression of huc expression in response to the availability of glycerol but not other carbon sources. Based on proteomic analyses and activity assays, mutation or knockdown of gylR leads to enhanced Huc production and activity. GylR and other key catabolite repressor proteins (Crp1, Crp2) do not directly bind the huc operon, indicating repression is mediated by unidentified transcription factors, with GylR acting as an upstream sensor. Here, we present data that suggests atmospheric H2 oxidation is regulated in response to organic carbon source availability through the process of catabolite repression. By identifying a key signal that prompts atmospheric H2 oxidation, these findings advance understanding of how aerobic bacteria adapt to changing environmental conditions and suggest that organic carbon levels are a key factor regulating the main sink of atmospheric H2 in soils globally. ImportanceSoil microorganisms collectively consume 70 million tonnes of atmospheric H2 a year, regulating atmospheric composition and climate change. In turn, consuming this dependable trace gas enables these microorganisms to survive even when their preferred organic energy sources are exhausted. Despite the importance of H2 consumption for soil biodiversity and atmospheric regulation, the signals and sensors that regulate this process remain to be understood. Here, we demonstrate that a model soil bacterium turns on the machinery required for atmospheric H2 consumption in direct response to being limited by organic carbon availability, through the process of catabolite repression. Specifically, in the absence of a sensor of the organic carbon source glycerol, a H2-consuming hydrogenase is highly expressed and active. These findings suggest that organic carbon levels have a major role in regulating trace gas oxidation, with implications for predicting how trace gas consumption and soil biodiversity respond to environmental change.

microbiology↗

bioRxiv2025

Integrated cross-sectoral surveillance of antimicrobial resistance genotypes and phenotypes across disparate reservoirs

Antimicrobial-resistant (AMR) bacteria and genes are continually exchanged among humans, animals, and environmental reservoirs. Disparate and siloed surveillance methods present a major challenge for tracing and disrupting AMR transmission, with clinical monitoring focusing on detecting specific pathogens or specific genes of interest, and environmental surveys often relying on inferences drawn from indicator organisms. Here, we demonstrate that, following sample-specific pre-processing, common surveillance approaches can be applied consistently to profile AMR abundance, distribution, and phenotypes across diverse reservoirs, including soil, sediment, water, wastewater, and faecal samples from both urban and agricultural settings. Across all sample types, three core methods provided complementary insights: (i) quantitative PCR (qPCR) arrays to measure multiple AMR genes, (ii) gene- and genome-centric metagenomics for comprehensive resistome profiling, and (iii) culture-based genomics with susceptibility testing to link genotypes to phenotypes. We applied this approach to profile 1,032 metagenome-assembled genomes, 66 bacterial isolate genomes, and 78 and 6,442 AMR genes/reference sequences via qPCR and metagenomics, respectively. This integrated framework revealed a moderate prevalence but high diversity of resistance mechanisms in both pathogens and non-pathogenic bacteria with potentially transmissible genes, with wastewater especially enriched in AMR genes. We detected mismatches between genotype and phenotype predictions and a prevalence of intermediate resistance phenotypes, highlighting how many mechanisms of environmental resistance remain poorly understood. Overall, this study demonstrates that unified field-leading surveillance methods can be extended beyond clinical contexts into diverse environmental and animal samples, while highlighting that multiple methods are needed to capture the diverse AMR genotypes and phenotypes in these settings to enable comprehensive monitoring and adaptive solutions to restrict transmission.

microbiology↗

bioRxiv2025

A Protein Antibiotic Inhibits the BAM Complex to Kill Without Cell Entry

Many antibiotics are ineffective against Gram-negative pathogens such as Pseudomonas aeruginosa because they cannot penetrate the bacterial outer membrane. Here, we show that protein antibiotics called L-type pyocins kill P. aeruginosa by inhibiting the {beta}-barrel assembly machinery (BAM) complex at the cell surface, halting outer-membrane protein assembly. Using single-particle cryo-electron microscopy, we show that L-type pyocins bind a surface-exposed region of BamA and deploy a C-terminal peptide that competitively inhibits the BAM complex, demonstrating that cell entry is not required for antibiotic activity. We combine genetics, multi-omics and cryo-electron tomography to show that BAM complex inhibition by L-type pyocins or the peptide antibiotic darobactin triggers a multifaceted transcriptomic, proteomic and morphological response. Despite this, BAM inhibition ultimately leads to a catastrophic loss of membrane integrity and cell death. These results validate BAM as a target for antibiotics that do not enter the cell and define an engineerable system for their development.

microbiology↗

bioRxiv2025

Hydrogenase-driven ATP synthesis from air

All cells require a continuous supply of the universal energy currency, adenosine triphosphate (ATP), to drive countless cellular reactions. The universally conserved F1Fo-ATP synthase regenerates ATP from ADP and Pi by harnessing a transmembrane electrochemical proton gradient (pmf). Bacteria have evolved a wide diversity of pmf-forming proteins to make ATP using light, organic, and inorganic energy sources. Recently, we proposed that many bacteria survive using atmospheric trace gases to produce ATP when limited for other energy sources. However, there is no direct proof that atmospheric energy sources are sufficient to generate pmf or drive ATP synthesis. Here, we show that the membrane-associated hydrogen:quinone oxidoreductase Huc from Mycobacterium smegmatis enables ATP synthesis from air. Purified Huc couples H2 oxidation to the reduction of various ubiquinone and menaquinone analogues. We designed and optimised a minimal respiratory chain in which Huc is reconstituted into liposomes with a pmf-generating terminal oxidase and the ATP-generating F1Fo-ATP synthase. Our experiments show that passive hydrogen exchange from air to solution is sufficient for the electron transfer and pmf generation required to accumulate ATP. Finally, by combining continuous culture bioenergetics measurements with theoretical calculations, we show this process is sufficient for mycobacteria to sustain pmf and ATP synthesis (two ATP molecules per H2 oxidised) for maintenance energy requirements during nutrient starvation. These findings prove that atmospheric energy sources are dependable lifeline substrates that enable continuous energy conservation during nutrient starvation. In addition, this work provides a new tool for ATP production in synthetic applications, which unlike other approaches is traceless without by-product accumulation.

microbiology↗

bioRxiv2024

De novo design of potent CRISPR-Cas13 inhibitors

CRISPR-Cas systems are transformative tools for gene editing which can be tuned or controlled by anti-CRISPRs (Acrs) - phage derived inhibitors that regulate CRISPR-Cas activity. However, Acrs that are capable of inhibiting biotechnologically relevant CRISPR systems are relatively rare and challenging to discover. To overcome this limitation, we describe a highly successful, rapid, and generalisable approach that leverages de novo protein design to develop new-to-nature proteins for controlling CRISPR-Cas activity. Using CRISPR-Cas13 as a representative example, we demonstrate that AI-designed anti-CRISPRs (AIcrs) are capable of highly potent and specific inhibition of CRISPR-Cas13 proteins. We present a comprehensive workflow for design validation and demonstrate AIcrs functionality in controlling CRISPR-Cas13 activity in bacteria. The ability to design bespoke inhibitors of Cas effectors will contribute to the ongoing development of CRISPR-Cas tools in diverse applications across research, medicine, agriculture, and microbiology.

biochemistry↗

bioRxiv2024

Inhibiting heme-piracy by pathogenic Escherichia coli using de novo-designed proteins

Iron is an essential nutrient for most bacteria and is often growth-limiting during infection, due to the host sequestering free iron as part of the innate immune response. To obtain the iron required for growth, many bacterial pathogens encode transporters capable of extracting the iron-containing cofactor heme directly from host proteins. Pathogenic E. coli and Shigella spp. produce the outer membrane transporter ChuA, which binds host hemoglobin and extracts its heme cofactor, before importing heme into the cell. Heme extraction by ChuA is a dynamic process, with the transporter capable of rapidly extracting heme from hemoglobin in the absence of an external energy source, without forming a stable ChuA-hemoglobin complex. In this work, we utilise a combination of structural modelling, Cryo-EM, X-ray crystallography, mutagenesis, and phenotypic analysis to understand the mechanistic detail of this process. Based on this understanding we utilise artificial intelligence-based protein design to create binders capable of inhibiting E. coli growth by blocking hemoglobin binding to ChuA. By screening a limited number of these designs, we identify several binders that inhibit E. coli growth at low nanomolar concentrations, without experimental optimisation. We determine the structure of a subset of these binders, alone and in complex with ChuA, demonstrating that they closely match the computational design. This work demonstrates the utility of de novo-designed proteins for inhibiting bacterial nutrient uptake and uses a workflow that could be applied to integral membrane proteins in other organisms.

biochemistry↗

bioRxiv2024

Methanogenesis inhibition remodels microbial fermentation and stimulates acetogenesis in ruminants

Rumen microbiota enable ruminants to grow on fibrous plant materials but also produce methane, driving 5% of global greenhouse gas emissions and leading to a loss of gross energy content. Methanogenesis inhibitors such as 3-nitrooxypropanol (3-NOP) decrease methane emissions in ruminants when supplemented in feed. Yet we lack a system-wide, species-resolved understanding of how the rumen microbiota remodels following inhibition and how this influences animal production. Here, we conducted a large-scale trial with 51 dairy calves to analyse microbiota responses to 3-NOP, pairing host performance, emissions, and nutritional profiles with genome-resolved metagenomic and metatranscriptomic data. 3-NOP supplementation decreased methane emissions by an average of 62%, modulated short-chain fatty acid and H2 levels, and did not affect dietary intake or animal performance. We created a rumen microbial genome catalogue with an unprecedented mapping rate. We observed a strong reduction of methanogens and stimulation of reductive acetogens, primarily novel uncultivated lineages such as Candidatus Faecousia. However, there was a shift in major fermentative communities away from acetate production in response to hydrogen gas accumulation. Thus, the divergent responses of the fermentative and hydrogenotrophic communities limit potential productivity gains from methane reduction. Reporting one of the largest reductions in methane emissions in a field trial to date, this study links ruminant greenhouse gas emissions and productivity to specific microbial species. These findings also emphasise the importance of microbiota-wide analysis for optimising methane mitigation strategies and identify promising strategies to simultaneously reduce emissions while increasing animal production. Significance StatementOne strategy to increase the sustainability and productivity of livestock production is to modulate ruminant microbiota to produce absorbable nutrients rather than the potent greenhouse gas methane. Previous studies show supplementing feed with methanogenesis inhibitors such as 3-nitrooxypropanol reduces methane emissions, but also leads to inconsistent productivity gains. Here we report a definitive field trial, combining animal data, meta-omics, and structural modelling, to resolve the key microbes and pathways controlling nutrient and methane production in ruminants. We show that shifts in composition and gene expression of hydrogen-cycling microbes reduce emissions but limit productivity gains. These findings offer insights at unprecedented resolution, while the data and analytical framework provide valuable resources to develop solutions to enhance livestock productivity and sustainability.

microbiology↗

bioRxiv2024

High-affinity PQQ import is widespread in Gram-negative bacteria

PQQ is a soluble redox cofactor used by diverse bacteria to oxidise fuel compounds as a source of electrons for the respiratory chain. Many Gram-negative bacteria that encode PQQ-dependent enzymes do not possess the biosynthetic machinery for its production and instead obtain it from the environment. To achieve this the bacterium Escherichia coli uses the TonB-dependent transporter PqqU as a high-affinity PQQ importer, allowing it to use PQQ at an external concentration as low as 1 nM. Here, we show that PqqU achieves this by binding PQQ with a very high affinity. Using cryo-electron microscopy we determine the structure of the PqqU-PQQ complex at a resolution of 1.99 [A], revealing that the extracellular loops of PqqU undergo significant conformational changes upon PQQ binding, which captures the cofactor in an internal cavity. This cavity likely facilitates an airlock-style gating mechanism that prevents non-specific import through PqqU. Using structural modelling we show that the change in PqqU structure upon PQQ binding precludes the binding of bacteriophage, which targets it as a cell surface receptor. Guided by the PqqU-PQQ complex structure we use phenotypic analysis to identify the amino acids essential for PQQ import and leverage this information to map the presence of PqqU across Gram-negative bacteria. This reveals that PqqU is encoded by Gram-negative bacteria from at least 22 phyla from diverse habitats, including those found in aquatic, soil, host-associated, and extreme environments. This indicates that PQQ is a ubiquitous nutrient in many environments, and an important cofactor for bacteria that adopt diverse lifestyles and metabolic strategies. Significance StatementMany enzymes form complexes with molecules called cofactors to perform their function. PQQ is a cofactor used by bacterial enzymes that provide energy by breaking down food molecules. While some bacteria make their own PQQ, other bacteria use the transport protein PqqU to bind PQQ from the environment and import it into their cells. We show that PqqU binds PQQ very tightly, allowing bacteria to acquire it at very low concentrations. Using cryo-electron microscopy we image the PqqU-PQQ complex on an atomic level, revealing how PQQ is bound so tightly. Using this the information to analyse microbial genomes, we show that PQQ scavenging is employed by diverse bacteria, implying that PQQ is an important common good of diverse microbiomes.

biochemistry↗

bioRxiv2024

Obtaining high-resolution cryo-EM structures using a common LaB6, 120-keV electron microscope equipped with a sub 200-keV optimised direct electron detector.

Cryo-electron microscopy (Cryo-EM) single particle analysis (SPA) has become a major structural biology technique in recent years. High-resolution cryo-EM typically requires higher voltage cryo-TEMs with coherent FEG sources, stable columns, autoloader systems and direct electron detectors. These setups are specialised for Cryo-EM work and are expensive to establish and maintain. More recently the concept of using 100-keV cryo-TEMs has been introduced as a way to make cryo-EM more affordable and hence accessible to a larger group of researchers. So far, the implementation of these 100-keV cryo-TEMs have relied on specialised microscopes with FEG sources as well as more stable optics than usually present on the common 120-keV TEMs. We here explored whether a standard 120-keV TEM, commonly available at many laboratories worldwide, can be upgraded with a direct electron detector and its suitability for high-resolution cryo-EM using a standard side entry cryo-holder. Using this imaging configuration, we were successful in achieving a 2.65[A] reconstruction for standard apoferritin. We were also able to resolve a more challenging small 64kDa protein haemoglobin to 4.33[A]. Furthermore, we were able to solve an asymmetric 153 kDa membrane protein GPCR (M4 muscarinic acid receptor) to a resolution of 4.4[A]. Importantly, all these results were achieved using a standard automated data collection routine implemented through SerialEM, making it feasible to collect large cryo-EM data sets with a side entry cryo-holder. These results showcase a potentially widely accessible solution to obtaining interpretable cryo-EM structures. Furthermore, we envisage that this imaging configuration gives an option for many EM facilities and laboratories to set up a high-quality cryo-EM SPA sample screening capability without the need to procure costly specialised Cryo-TEMs. This could help to considerably lower the economic entry barrier for cryo-EM SPA and contribute to the "democratisation" of cryo-EM.

biophysics↗

bioRxiv2024

Quinone extraction drives atmospheric carbon monoxide oxidation in bacteria

Diverse bacteria and archaea use atmospheric carbon monoxide (CO) as an energy source during long-term survival. This process enhances the biodiversity of soil and marine ecosystems globally and removes 250 million tonnes of a toxic, climate-relevant pollutant from the atmosphere each year. Bacteria use [MoCu]-carbon monoxide dehydrogenases (Mo-CODH) to convert CO to carbon dioxide, then transfer the liberated high-energy electrons to the aerobic respiratory chain. However, given no high-affinity Mo-CODH has been purified, it is unknown how these enzymes oxidise CO at low concentrations and interact with the respiratory chain. Here we resolve these knowledge gaps by analysing Mo-CODH (CoxSML) and its hypothetical partner CoxG from Mycobacterium smegmatis. Kinetic and electrochemical analyses show purified Mo-CODH is a highly active high-affinity enzyme (Km = 139 nM, kcat = 54.2 s-1). Based on its 1.85 [A] resolution cryoEM structure, Mo-CODH forms a CoxSML homodimer similar to characterised low-affinity homologs, but has distinct active site coordination and narrower gas channels that may modulate affinity. We provide structural, biochemical, and genetic evidence that Mo-CODH transfers CO-derived electrons to the aerobic respiratory chain via the membrane-bound menaquinone-binding protein CoxG. Consistently, CoxG is required for CO-driven respiration, extracts menaquinone from mycobacterial membranes, and binds quinones in a hydrophobic pocket. Finally, we show that Mo-CODH and CoxG genetically and structurally associate in diverse bacteria and archaea. These findings reveal the basis of a biogeochemically and ecologically important process, while demonstrating that the newly discovered process of long-range quinone transport is a general mechanism of energy conservation, which convergently evolved on multiple occasions.

biochemistry↗

bioRxiv2023

Metabolism of ʟ -arabinose converges with virulence regulation to promote enteric pathogen fitness

Virulence and metabolism are often interlinked to control the expression of essential colonisation factors in response to host-associated signals. Here, we identified a novel transporter of the dietary monosaccharide -arabinose that is widely encoded by the zoonotic pathogen enterohaemorrhagic Escherichia coli (EHEC), required for full competitive fitness in the mouse gut and highly expressed during human infection. Accordingly, we discovered that -arabinose induces expression of the EHEC type 3 secretion system, enhancing its ability to attach to host cells, and that the underlying mechanism is dependent on products of its catabolism rather than the sensing of -arabinose as a signal. Finally, using the murine pathogen Citrobacter rodentium, we show that -arabinose metabolism provides a fitness benefit during enteric infection via virulence factor regulation, as opposed to supporting pathogen growth. This study describes an intrinsic mechanism of integrating central sugar metabolism with virulence regulation and highlights the unexpected impact that nutrient utilisation can have in enteric pathogens.

microbiology↗

bioRxiv2023

An obligate aerobe adapts to hypoxia by hybridising fermentation with carbon storage

In soil ecosystems, obligately aerobic bacteria survive oxygen deprivation (hypoxia) by entering non-replicative persistent states. Little is known about how these bacteria rewire their metabolism to stay viable in these states. The model obligate aerobe Mycobacterium smegmatis maintains redox homeostasis during hypoxia by mediating fermentative hydrogen production. However, the fate of organic carbon during fermentation, and the associated remodeling of carbon metabolism, is unresolved. Here we systematically profiled the metabolism of M. smegmatis during aerobic growth, hypoxic persistence, and the transition between these states. Using differential isotope labelling, and paired metabolomics and proteomics, we observed rerouting of central carbon metabolism through the pentose phosphate pathway and Entner-Doudoroff pathway during hypoxia. We show that M. smegmatis excretes high levels of hydrogen concurrently with upregulating triacylglyceride synthases and accumulating glycerides as carbon stores. Using electron cryotomography (cryo-ET), we observed the presence of large spheroid structures consistent with the appearance of lipid droplets. Thus, in contrast to obligately and facultative anaerobic fermentative bacteria, M. smegmatis stores rather than excretes organic carbon during hypoxia. This novel hybrid metabolism likely provides a competitive advantage in resource-variable environments by allowing M. smegmatis to simultaneously dispose excess reductant during hypoxia and maintain carbon stores to rapidly resume growth upon reoxygenation.

microbiology↗

bioRxiv2022

Atmospheric hydrogen oxidation extends to the domain archaea

Diverse aerobic bacteria use atmospheric hydrogen (H2) and carbon monoxide (CO) as energy sources to support growth and survival. Though recently discovered, trace gas oxidation is now recognised as a globally significant process that serves as the main sink in the biogeochemical H2 cycle and sustains microbial biodiversity in oligotrophic ecosystems. While trace gas oxidation has been reported in nine phyla of bacteria, it was not known whether archaea also use atmospheric H2. Here we show that a thermoacidophilic archaeon, Acidianus brierleyi (Thermoproteota), constitutively consumes H2 and CO to sub-atmospheric levels. Oxidation occurred during both growth and survival across a wide range of temperatures (10 to 70{degrees}C). Genomic analysis demonstrated that A. brierleyi encodes a canonical carbon monoxide dehydrogenase and, unexpectedly, four distinct [NiFe]-hydrogenases from subgroups not known to mediate aerobic H2 uptake. Quantitative proteomic analyses showed that A. brierleyi differentially produced these enzymes in response to electron donor and acceptor availability. A previously unidentified group 1 [NiFe]-hydrogenase, with a unique genetic arrangement, is constitutively expressed and upregulated during stationary phase and aerobic hydrogenotrophic growth. Another archaeon, Metallosphaera sedula, was also found to oxidize atmospheric H2. These results suggest that trace gas oxidation is a common trait of aerobic archaea, which likely plays a role in their survival and niche expansion, including during dispersal through temperate environments. These findings also demonstrate that atmospheric H2 consumption is a cross-domain phenomenon, suggesting an ancient origin of this trait, and identify previously unknown microbial and enzymatic sinks of atmospheric H2 and CO.

microbiology↗