Biology subjects

Higginbottom, S. K.

Publications and source records attributed to Higginbottom, S. K..

18 recordsLinked to original sources

bioRxiv2026

Tracking gene flow in the gut microbiome reveals phage activation and host-range expansion in response to diet

Horizontal gene transfer (HGT) drives microbial evolution, but what determines when genes move between species within complex communities remains unclear. We developed a junction-resolved metagenomic framework using short-read sequencing to distinguish mobile genetic element activation from de novo transfer and track both longitudinally. In a defined human gut community colonizing mice, mobile element activation was widespread but successful transfer was rare. A prophage carried by invading Phocaeicola vulgatus transferred reproducibly to resident species when the community resisted invasion, but not when dietary perturbation allowed the invader to expand. Transfer required prophage activation and persistence of a permissive recipient and coincided with diversification of a phage receptor-binding protein. Dietary polysaccharides reproduced prophage-associated fitness costs in vitro, and rhamnogalacturonan I induced cell lysis consistent with productive prophage activation. The framework also detected interspecies transfer retrospectively in longitudinal human metagenomes. Thus, diet and community ecology can redirect gene flow through the gut microbiome.

microbiology↗

bioRxiv2026

Functional Divergence in Gut Clostridia from Hadza Hunter-Gatherers Reveals Diet-Adapted Metabolic Machinery That Shapes Gut Ecology

Lifestyle changes associated with industrialization, including shifts in diet, are linked to extensive remodeling of the human gut microbiome. Investigating the gut microbiomes of traditional populations living non-industrialized lifestyles, such as the Hadza hunter-gatherers of Tanzania, therefore offers insight into bacterial taxa and functions depleted in industrialized microbiomes. Despite pronounced compositional differences that correspond to lifestyle, many commensal gut species are shared between industrialized and non-industrialized populations, raising a fundamental question of whether and how their functional repertoires diverge across contrasting gut environments. Here, by integrating strain-resolved metagenomics with culturomics and in vivo models, we identify phylogenetic and functional divergence within prevalent gut species shared between Hadza and industrialized populations, particularly among members of the Clostridia class. Dorea longicatena, prevalent across diverse human populations independent of lifestyle, is a poorly characterized member of Clostridia. Using strains of this D. longicatena isolated from either Hadza or industrialized human stool samples we uncover metabolic pathways selectively enriched in Hadza strains, including machinery for utilizing plant-derived pectic polysaccharides. We further demonstrate that this Hadza-enriched polysaccharide-utilization machinery can be leveraged with its cognate dietary substrate to promote D. longicatena colonization within a model industrialized microbiota, reshaping intestinal and portal-vein metabolite profiles. Together, our findings reveal substantial functional divergence within gut bacterial species shared across lifestyles, providing a framework for identifying diet-responsive strains and pathways that may complement ecological and metabolic functions underrepresented in industrialized microbiotas.

microbiology↗

bioRxiv2026

Ecology and engineering to modify the bile acid output of a defined microbial community

The bile acid pool, which is synthesized collaboratively by the host and its microbiome, impacts metabolism, immunity, and disease risk. Targeted microbiome interventions could in principle reshape the bile acid pool for therapeutic benefit, but practical strategies remain elusive. In the course of screening a complex defined community for metabolic phenotypes by dropping out individual strains, we observed that several of the single-strain dropout communities had markedly increased deoxycholic and lithocholic acid levels and a larger bile acid pool. In each of these communities, a second strain--Lactobacillus plantarum--had bloomed. The bile salt hydrolase activity of L. plantarum was necessary and sufficient to expand the size of the bile acid pool. An engineered community in which the bsh gene is overexpressed in multiple Lactobacillus strains confers on mice increased levels of secondary bile acid levels and a larger pool size. By overexpressing a different pair of bile acid metabolic genes in multiple strains of Lactobacillus--7- and 7{beta}-hydroxysteroid dehydrogenase--we changed the composition of the bile acid pool, enlarging it and redirecting it toward ursodeoxycholic acid. Together, these results demonstrate that fine details of the microbiomes strain composition can have a substantial effect on bile acid metabolism, and that rational manipulation of the microbiome can alter the size and composition of the bile acid pool.

microbiology↗

bioRxiv2026

A single-strain dropout screen reveals mechanistic links between microbial ecology and metabolism

The complexity of the gut microbiome has made it challenging to define the role of individual species in community-level function. Here, we constructed 56 single-strain dropout variants of a defined 118-member community and used each one to colonize a group of germ-free mice. In many cases, removing a single strain triggered a large reordering of a small group of species, which in turn altered the communitys metabolic output. En bloc removal of the eight-strain acetogen compartment markedly reduced acetate production and caused intestinal H2 accumulation and bloating; a specific subset of four acetogens was sufficient to relieve bloating and restore acetate production. Together, these data show that small disturbances in community composition can trigger a confined ecological reorganization with a large chemical phenotype, and they reveal novel strategies for engineering communities with altered metabolic output.

microbiology↗

bioRxiv2026

Indole-3-acetic acid production is rare among gut bacteria and reflects OFOR-driven amino acid oxidation in acetogens

Indole-3-acetic acid (IAA) is a tryptophan-derived gut microbial metabolite with reported anti-inflammatory activities, but the organisms and anaerobic pathways that support robust production remain unclear. Screening 206 human gut bacterial isolates by LC-MS revealed that IAA production is rare: only five strains exceeded the limit of quantitation, and high-capacity production was confined to the acetogens Blautia hydrogenotrophica and Intestinibacter bartlettii. Across growth conditions, IAA was a minor product that rose alongside carbohydrate-sensitive, OFOR-linked catabolism of multiple amino acids, generating abundant branched-chain and aromatic organic acids. In gnotobiotic mice mono-colonized with I. bartlettii, these metabolites were produced in vivo but showed distinct host handling, with branched-chain fatty acids largely extracted between portal and peripheral plasma, whereas aromatic acids and their glycine conjugates appeared in plasma and urine. Genomic analyses and heterologous enzyme assays identified expanded repertoires of 2-oxoacid:ferredoxin oxidoreductases (OFORs) with activities spanning pyruvate/oxaloacetate, branched-chain, and aromatic 2-oxoacids, including indolepyruvate conversion to indoleacetyl-CoA, a putative intermediate en route to IAA. Finally, position-specific 13C tracing showed that CO2 released during amino acid oxidation is reassimilated into acetate via reductive acetogenesis, indicating that gut acetogens can maintain redox balance without fermenting partner strains. Together, these findings show that high IAA output is restricted to select gut acetogens and linked to a broader OFOR-driven anaerobic metabolism that generates additional metabolites that are absorbed by the host.

microbiology↗

bioRxiv2024

Gut microbiota gate host exposure to metabolites from dietary Solanums

Dietary plants are molecularly rich but the fates of these compounds post-ingestion and their implications for human health are largely unknown. Here, we systematically characterized the major chemical contributions of widely consumed Solanum species (nightshades) to the human metabolome. Using untargeted metabolomics, we found that a series of steroidal alkaloids resulting from glycoalkaloids tomatine, solanine, and chaconine are dominant diet-derived compounds in systemic circulation following ingestion of tomato and potato. By comparing serum and tissue metabolomes of colonized and microbiome-depleted mice, we determined that the gut microbiota modifies these compounds extensively, altering their absorption and gating host exposure. By screening the metabolic products in human urine and stool samples, we established that steroidal glycoalkaloid metabolism varies inter-individually in a population. Furthermore, using a collection of representative human commensal type strains, we found that a limited set of strains is responsible for steroidal glycoalkaloid metabolism, with the chemical output of a community determined by its strain-level composition. These findings enabled the rational design of complex synthetic microbial communities that controlled host exposure to steroidal alkaloid metabolites in vivo. Importantly, microbial metabolism of Solanum metabolites alters their acetylcholinesterase inhibition in vitro and gut motility in vivo. Our study provides insights into the molecular mechanisms of a diet-microbiome interactions and the effects of dietary metabolites on host physiology.

microbiology↗

bioRxiv2024

High-resolution lineage tracking of within-host evolution and strain transmission in a human gut symbiont across ecological scales

Gut bacteria rapidly evolve in vivo, but their long-term success requires dispersal across hosts. Here, we quantify this interplay by tracking >50,000 genomically barcoded lineages of the prevalent commensal Bacteroides thetaiotaomicron (Bt) among co-housed mice. We find that adaptive mutations rapidly spread between hosts, overcoming the natural colonization resistance of resident Bt strains. Daily transmission rates varied >10-fold across hosts, but shared selection pressures drove predictable engraftment of specific lineages over time. The addition of a 49-species community shifted the adaptive landscape relative to mono-colonized Bt without slowing the rate of evolution, and reduced transmission while still allowing specific mutants to engraft. Whole-genome sequencing uncovered diverse modes of adaptation involving complex carbohydrate metabolism. Complementary in vitro evolution across 29 carbon sources revealed variable overlap with in vivo selection pressures, potentially reflecting synergistic and antagonistic pleiotropies. These results show how high-resolution lineage tracking enables quantification of commensal evolution across ecological scales.

evolutionary biology↗

bioRxiv2023

A mutant fitness compendium in Bifidobacteria reveals molecular determinants of colonization and host-microbe interactions

Bifidobacteria commonly represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest as a probiotic therapy, predicting the nutritional requirements and health-promoting effects of Bifidobacteria is challenging due to major knowledge gaps. To overcome these deficiencies, we used large-scale genetics to create a compendium of mutant fitness in Bifidobacterium breve (Bb). We generated a high density, randomly barcoded transposon insertion pool in Bb, and used this pool to determine Bb fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. To enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1462 genes. We leveraged these tools to improve models of metabolic pathways, reveal unexpected host- and diet-specific requirements for colonization, and connect the production of immunomodulatory molecules to growth benefits. These resources will greatly reduce the barrier to future investigations of this important beneficial microbe.

microbiology↗

bioRxiv2023

Hadza Prevotella Require Diet-derived Microbiota Accessible Carbohydrates to Persist in Mice.

Industrialization has transformed the gut microbiota, reducing the prevalence of Prevotella relative to Bacteroides. Here, we isolate Bacteroides and Prevotella strains from the microbiota of Hadza hunter-gatherers of Tanzania, a population with high levels of Prevotella. We demonstrate that plant-derived microbiota-accessible carbohydrates (MACs) are required for persistence of Prevotella copri but not Bacteroides thetaiotaomicron in vivo. Differences in carbohydrate metabolism gene content, expression, and in vitro growth reveal that Hadza Prevotella strains specialize in degrading plant carbohydrates, while Hadza Bacteroides isolates use both plant and host-derived carbohydrates, a difference mirrored in Bacteroides from non-Hadza populations. When competing directly, P. copri requires plant-derived MACs to maintain colonization in the presence of B. thetaiotaomicron, as a no MAC diet eliminates P. copri colonization. Prevotellas reliance on plant-derived MACs and Bacteroides ability to use host mucus carbohydrates could explain the reduced prevalence of Prevotella in populations consuming a low-MAC, industrialized diet. Statement on work with indigenous communitiesIn order to acquire scientific knowledge that accurately represents all human populations, rather than only reflecting and benefiting those in industrialized nations, it is necessary to involve indigenous populations in research in a legal, ethical, and non-exploitative manner (Abdill et al., 2022; Green et al., 2020). Here, we isolated live bacterial strains from anonymized fecal samples collected from Hadza hunter-gatherers in 2013/2014 (Fragiadakis et al., 2019; Merrill et al., 2022; Smits et al., 2017). Samples were collected with permission from the Tanzanian government, National Institute of Medical Research (MR/53i 100/83, NIMR/HQ/R.8a/Vol.IX/1542), the Tanzania Commission for Science and Technology, and with aid from Tanzanian scientists. A material transfer agreement with the National Institute for Medical Research in Tanzania specifies that collected samples are solely to be used for academic purposes. For more information on the consent practices followed, and our ongoing work to communicate the results of these projects to the Hadza, please see (Merrill et al., 2022; Olm et al., 2022).

microbiology↗

bioRxiv2022

Metabolic diversity in commensal protists regulates intestinal immunity and trans-kingdom competition

The microbiota influences intestinal health and physiology, yet the contributions of commensal protists to the gut environment have been largely overlooked. Here, we identified several new rodent- and human-associated parabasalid protists. Genomic and metabolomic analyses of murine parabasalids from the genus Tritrichomonas revealed species-level differences in the excretion of the metabolite succinate. This metabolic dissimilarity results in distinct small intestinal immune responses during protist colonization. Metabolic differences between Tritrichomonas species also determine their ecological niche within the microbiota. By manipulating dietary fibers and developing in vitro protist culture, we show that different parabasalid species preferentially rely on dietary polysaccharides or mucus glycans. These polysaccharide preferences create trans-kingdom competition with specific commensal bacteria, which affects intestinal immunity in a diet-dependent manner. Our findings reveal unappreciated diversity in commensal parabasalids, elucidate differences in commensal protist metabolism, and suggest how dietary interventions could regulate their impact on gut health.

microbiology↗

bioRxiv2022

Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome

The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by focusing on a functional unit within the community, the metabolic niche that controls bile acid 7-dehydroxylation. By constructing variants of a complex defined community in which we drop out strains that occupy this niche, we explore how interactions within and between niches shape community-level metabolism. Omitting both members of the niche, Clostridium scindens (Cs) and Clostridium hylemonae (Ch), eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains go up or down in relative abundance by >100-fold, while the remaining strains are largely unaffected. In single-strain dropout communities (i.e., a strain swap within the niche), Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, the effect on strains in other niches differs markedly: Clostridium sporogenes increases >1000-fold in the {Delta}Cs but not {Delta}Ch dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype. Mice colonized by the {Delta}Cs{Delta}Ch community show decreased liver steatosis relative to those colonized by the {Delta}Ch community, demonstrating that a single strain from the microbiome can have a substantive impact on host physiology. Our work opens the door to the mechanistic studies of the role of an individual strain on community ecology and host physiology.

microbiology↗

bioRxiv2022

Butyrate differentiates permissiveness to Clostridioides difficile infection and influences growth of diverse C. difficile isolates

A disrupted "dysbiotic" gut microbiome engenders susceptibility to the diarrheal pathogen Clostridioides difficile by impacting the metabolic milieu of the gut. Diet, in particular the microbiota accessible carbohydrates (MACs) found in dietary fiber, is one of the most powerful ways to affect the composition and metabolic output of the gut microbiome. As such, diet is a powerful tool for understanding the biology of C. difficile and for developing alternative approaches for coping with this pathogen. One prominent class of metabolites produced by the gut microbiome are short chain fatty acids (SCFAs), the major metabolic end products of MAC metabolism. SCFAs are known decrease the fitness of C. difficile in vitro and that high intestinal SCFA concentrations are associated with reduced fitness of C. difficile in animal models of C. difficile infection (CDI). Here, we use controlled dietary conditions (8 diets that differ only by MAC composition) to show that C. difficile fitness is most consistently impacted by butyrate, rather than the other two prominent SCFAs (acetate and propionate), during murine model CDI. We similarly show that butyrate concentrations are lower in fecal samples from humans with CDI relative to healthy controls. Finally, we demonstrate that butyrate impacts growth in diverse C. difficile isolates. These findings provide a foundation for future work which will dissect how butyrate directly impacts C. difficile fitness and will lead to the development of diverse approaches distinct from antibiotics or fecal transplant, such as dietary interventions, for mitigating CDI in at-risk human populations. IMPORTANCEClostridioides difficile is a leading cause of infectious diarrhea in humans and it imposes a tremendous burden on the healthcare system. Current treatments for C. difficile infection (CDI) include antibiotics and fecal microbiota transplant, which contribute to recurrent CDIs and face major regulatory hurdles, respectively. Therefore, there is an ongoing need to develop new ways to cope with CDI. Notably, a disrupted "dysbiotic" gut microbiota is the primary risk factor for CDI but we incompletely understand how a healthy microbiota resists CDI. Here, we show that a specific molecule produced by the gut microbiota, butyrate, is negatively associated with C. difficile burdens in humans and in a mouse model of CDI and that butyrate impedes the growth of diverse C. difficile strains in pure culture. These findings help to build a foundation for designing alternative, possibly diet-based, strategies for mitigating CDI in humans.

microbiology↗

bioRxiv2021

A metabolomics pipeline enables mechanistic interrogation of the gut microbiome

Gut microbes modulate host phenotypes and are associated with numerous health effects in humans, ranging from cancer immunotherapy response to metabolic disease and obesity. However, difficulty in accurate and high-throughput functional analysis of human gut microbes has hindered defining mechanistic connections between individual microbial strains and host phenotypes. One key way the gut microbiome influences host physiology is through the production of small molecules1-3, yet progress in elucidating this chemical interplay has been hindered by limited tools calibrated to detect products of anaerobic biochemistry in the gut. Here we construct a microbiome-focused, integrated mass-spectrometry pipeline to accelerate the identification of microbiota-dependent metabolites (MDMs) in diverse sample types. We report the metabolic profiles of 178 gut microbe strains using our library of 833 metabolites. Leveraging this metabolomics resource we establish deviations in the relationships between phylogeny and metabolism, use machine learning to discover novel metabolism in Bacteroides, and employ comparative genomics-based discovery of candidate biochemical pathways. MDMs can be detected in diverse biofluids in gnotobiotic and conventional mice and traced back to corresponding metabolomic profiles of cultured bacteria. Collectively, our microbiome-focused metabolomics pipeline and interactive metabolomics profile explorer are a powerful tool for characterizing microbe and microbe-host interactions.

microbiology↗

bioRxiv2020

Quantifying the interplay between rapid bacterial evolution within the mouse intestine and transmission between hosts

Due to limitations on high-resolution strain tracking, selection dynamics during gut-microbiota colonization and transmission between hosts remain mostly mysterious. Here, we introduced hundreds of barcoded Escherichia coli strains into germ-free mice and quantified strain-level dynamics and metagenomic changes. Mutants involved in motility and utilization of abundant metabolites were reproducibly selected within days. Even with rapid selection, coprophagy enforced similar barcode distributions across co-housed mice. Whole-genome sequencing of hundreds of isolates quantified evolutionary dynamics and revealed linked alleles. A population-genetics model predicted substantial fitness advantages for certain mutants and that migration accounted for ~10% of the resident microbiota each day. Treatment with ciprofloxacin demonstrated the interplay between selection and transmission. While initial colonization was mostly uniform, in two mice a bottleneck reduced diversity and selected for ciprofloxacin resistance in the absence of drug. These findings highlight the interplay between environmental transmission and rapid, deterministic selection during evolution of the intestinal microbiota.

microbiology↗

bioRxiv2020

High-throughput cultivation of stable, diverse, fecal-derived microbial communities to model the intestinal microbiota

SummaryMechanistic understanding of the impacts of the gut microbiota on human health has been hampered by limited throughput in animal models. To enable systematic interrogation of gut-relevant microbial communities, here we generated hundreds of in vitro communities cultured from diverse stool samples in various media. Species composition revealed stool-derived communities that are phylogenetically complex, diverse, stable, and highly reproducible. Community membership depended on both medium and initial inoculum, with certain media preserving inoculum compositions. Different inocula yielded different community compositions, indicating their potential for personalized therapeutics. Communities were robust to freezing and large-volume culturing, enabling future translational applications. Defined communities were generated from isolates and reconstituted growth and composition similar to those of communities derived from stool inocula. Finally, in vitro experiments probing the response to ciprofloxacin successfully predicted many changes observed in vivo, including the resilience and sensitivity of each Bacteroides species. Thus, stool-derived in vitro communities constitute a powerful resource for microbiota research.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2019

Electron transfer complexes in the gut dictate high abundance circulating metabolites

It has long been known that proteolytic Clostridia obtain their energy by coupling oxidative and reductive pathways for amino acid metabolism - the Stickland reaction1. The oxidation of one amino acid is coupled with reduction of another, yielding energy in the former step and re-achieving redox balance with the latter. Here, we find that the gut bacterium, Clostridium sporogenes metabolizes amino acids through reductive pathways to produce metabolites that circulate within the host. Measurements in vitro indicate that reductive Stickland pathways are coupled to ATP formation, revealing their role in energy capture by gut bacteria. By probing the genetics of C. sporogenes, we find that the Rnf complex is involved in reductive amino acid metabolism. Rnf complex mutants are attenuated for growth in the mouse gut, demonstrating the importance of energy capture during reductive metabolism for gut colonization. Our findings reveal that the production of high-abundance molecules by a commensal bacterium within the host gut is linked to an energy yielding redox process.

microbiology↗

bioRxiv2019

Recovery of the gut microbiota after antibiotics depends on host diet and environmental reservoirs

That antibiotics alter microbiota composition and increase infection susceptibility is well known, but their generalizable effects on the gut commensal community and dependence on environmental variables remain open questions. Here, we systematically compared antibiotic responses in gnotobiotic and conventional mice across antibiotics, microbiotas, diets, and housing status. We identify remarkable resilience, whereby a humanized microbiota recovers before drug administration ends, with transient dominance of resistant Bacteroides and taxa-asymmetric reduction in diversity. In other cases, in vitro sensitivities were not predictive of in vivo responses, underscoring the significance of host and community contexts. A fiber-deficient diet exacerbated collapse of the microbiota and delayed recovery, despite the presence of a similar core community across diets at the point of maximal disturbance. Resilience to a second ciprofloxacin treatment was observed via response reprogramming, in which species replacement after ciprofloxacin treatment established resilience to a second treatment, and also through cross housing transmission. Single-housing drastically disrupted recovery, highlighting the importance of environmental microbial reservoirs and suggesting sanitation may exacerbate the duration of antibiotic-mediated disruption. Our findings highlight the ability of the commensal microbiota to deterministically adapt to large perturbations, and the translational potential for modulating diet, sanitation, and microbiota composition during antibiotics.

microbiology↗

bioRxiv2019

A metabolic pathway for glucosinolate activation by the human gut symbiont Bacteroides thetaiotaomicron

Diet is the largest source of plant-derived metabolites that influence human health. The gut microbiota can metabolize these molecules, altering their biological function. However, little is known about the gut bacterial pathways that process plant-derived molecules. Glucosinolates are well-known metabolites in brassica vegetables and metabolic precursors to cancer-preventive isothiocyanates. Here, we identify a genetic and biochemical basis for isothiocyanate formation by Bacteroides thetaiotaomicron, a prominent gut commensal species. Using a genome-wide transposon insertion screen, we identified an operon required for glucosinolate metabolism in B. thetaiotaomicron. Expression of BT2159-BT2156 in a non-metabolizing relative, Bacteroides fragilis, resulted in gain of glucosinolate metabolism. We show that isothiocyanate formation requires the action of BT2158 and either BT2156 or BT2157 in vitro. Monocolonization of mice with mutant Bt{Delta}2157 showed reduced isothiocyanate production in the gastrointestinal tract. These data provide insight into the mechanisms by which a common gut bacterium processes an important dietary nutrient.

microbiology↗