Efflux pumps play a key role in both intrinsic and acquired antibiotic resistance in Acinetobacter baumannii, yet their broader physiological roles remain unclear. Here, we investigated the transcriptomic and phenotypic responses of A. baumannii ATCC19606 mutants lacking the efflux pumps AdeAB, AdeIJ, or CraA under chloramphenicol stress. The deletion of craA resulted in a 32-fold reduction in chloramphenicol MIC, while ΔadeIJ showed a modest 4-fold decrease, and ΔadeAB had no effect on chloramphenicol susceptibility. Transcriptomic profiling revealed minimal alterations in ΔcraA, but notable transcriptional reprogramming in ΔadeAB and ΔadeIJ, including upregulation of ribosomal genes, iron-sulfur cluster biogenesis, aromatic compound catabolism, and amino acid transport systems. Under chloramphenicol stress, ΔadeAB exhibited metabolic remodeling, activating oxidative stress defenses and protein quality control pathways while repressing type VI secretion. Both ΔadeIJ and wild-type strains upregulated arginine and glutamate metabolism, likely contributing to redox balance. Phenotypically, the ΔadeIJ strain showed elevated basal and H₂O₂-induced ROS levels, as well as heightened sensitivity to nitrosative stress and Cu2+, which may suggest a role for AdeIJ in oxidative and metal stress responses. Despite increased adeAB and craA expression under chloramphenicol stress, only craA deletion significantly impaired resistance, highlighting its dominant role in chloramphenicol efflux. Collectively, our findings reveal efflux pump-specific and strain-specific adaptations to antibiotic stress, positioning CraA as a major determinant of chloramphenicol resistance, while RND transporters such as AdeABC and AdeIJK may also be associated with broader stress adaptation processes, including redox and metal homeostasis, in A. baumannii.IMPORTANCEEfflux pumps are key drivers of multidrug resistance in Acinetobacter baumannii, yet their broader roles in stress adaptation remain insufficiently understood. Here, we show that the loss of major efflux systems reshapes the transcriptomic and metabolic landscape under chloramphenicol stress, a condition that also imposes oxidative stress. In particular, the RND efflux pump AdeIJK may be linked to alterations in cellular responses to oxidative, nitrosative, and metal stress, although the mechanistic basis of this interplay remains to be further investigated. Overall, these findings suggest that efflux pumps may contribute to bacterial resilience beyond drug resistance, providing additional insight into efflux system hierarchy and functional redundancy in A. baumannii, and may inform future studies aimed at understanding persistence and efflux-mediated multidrug resistance mechanisms.
Natural, non-genetic variation among host cells can shape infection outcomes, yet its role in intracellular parasite dynamics remains poorly understood. Here, we show that variability in hemoglobin (Hb) content among individual red blood cells (RBCs) modulates nutrient consumption by Plasmodium falciparum, the most lethal malaria parasite. Using label-free quantitative-phase imaging (QPI), we measured cytosolic Hb in thousands of uninfected and infected RBCs across the 48-h asexual cycle. Hb content varied widely among uninfected cells, and after invasion, infected cells mirrored and amplified this distribution: variability increased from 17% in uninfected RBCs to >100% in schizonts. A minimal predator-prey model accurately reproduced the observed spread in parasite Hb consumption, showing that host variability alone can generate much of the diversity in parasite feeding rates traditionally attributed to parasite factors. These findings provide the first evidence that host phenotypic noise drives variation in Hb consumption among genetically identical parasites. Incorporating host-to-host variability into within-host models could improve predictions of parasite growth, refine the activation kinetics of Hb-dependent antimalarials, and reveal analogous principles in other intracellular infections where host variability and host-parasite crosstalk can shape pathogen outcomes. This systems-level perspective emphasizes the need to account for host heterogeneity in both experimental design and therapeutic strategies.IMPORTANCECell-to-cell phenotypic variability is a fundamental feature of living systems, yet its impact on host-pathogen interactions has been largely overlooked. Here, we show that natural, non-genetic variation in red blood cell hemoglobin strongly shapes how Plasmodium falciparum-the most lethal malaria parasite-consumes Hb inside host cells. Using quantitative imaging and a mathematical model, we demonstrate that host variability alone can explain much of the observed differences in parasite feeding rates. This perspective reframes parasite Hb consumption as an emergent property of host-parasite interactions. More broadly, it highlights how natural variability among host cells can influence the course of intracellular infections, with potential implications for predicting pathogen growth and optimizing drug treatment strategies.
The tripartite interplay among plants, bacteria, and fungal endophytes is crucial for maintaining host plant fitness. However, how Epichloë endophytes influence plant-associated bacterial communities in cool-season grasses, particularly in ecologically important yet understudied species, such as Achnatherum inebrians, remains unclear. Using phylogenetic molecular ecological network analysis, we determined that seed-borne (seed epiphytic) and phyllosphere bacterial communities of Epichloë-infected (EI) A. inebrians exhibited reduced network complexity compared to Epichloë-free (EF) plants. Across all samples, Proteobacteria and Firmicutes dominated the keystone taxa, with Pseudomonas (OTU744 and OTU8264) consistently identified as a hub genus in both seed-borne and phyllosphere networks. Culture-based analysis revealed that endophyte-infected plants had a significantly (P < 0.05) higher relative abundance of Pseudomonas and Bacillus than EF A. inebrians, especially Pseudomonas comprised 13, 35, and 33% of isolates from the seed, leaf, and rhizosphere of A. inebrians, respectively. To capture potential functional diversity, we selected two phylogenetically distant Pseudomonas strains from each of the three ecological niches for further analysis. Inoculation of A. inebrians seedlings with these strains consistently promoted plant growth, enhanced forage quality (total nitrogen content), and improved nutritional value (ether extract). Whole-genome sequencing combined core-genome phylogenetic tree of the six Pseudomonas strains and confirmed that five strains belong to P. atacamensis, whereas Pse19 was P. cucumis. Our findings reveal that Epichloë endophytes modulate bacterial network stability and enrich plant-associated Pseudomonas, which synergistically enhance host performance. Collectively, this study provides a mechanistic framework for manipulating keystone taxa and beneficial isolates to improve grass productivity in grassland agricultural ecosystems.IMPORTANCEAlthough the tripartite interplay between plants, bacteria, and fungal endophytes, such as Epichloë, is recognized as vital for host fitness, the specific mechanisms through which these endophytes shape associated bacterial communities, particularly in ecologically significant grasses, such as Achnatherum inebrians, remain poorly understood. This study provides crucial mechanistic insights by revealing that Epichloë endophytes reconfigure the structure and stability of both seed-borne and phyllosphere bacterial networks in A. inebrians, leading to reduced complexity but enrichment of specific keystone taxa. We identify Pseudomonas as a consistently dominant hub genus across these niches. Notably, functional validation shows that diverse Pseudomonas isolates, representative of those enriched by the endophyte, significantly enhance host plant growth, forage quality, and nutritional value. Our findings reveal a synergistic mechanism where Epichloë endophytes modulate bacterial network stability to favor beneficial Pseudomonas populations, collectively boosting host performance.
When subject to sublethal rifampicin stress, susceptible Escherichia coli cells quickly adapt to increase survival. We investigated whether the transcription factor network contributes to this adaptation. First, a responsive cohort of 709 genes diverged from the control, suggesting strong disturbance. While this was largely a direct effect of rifampicin, we show evidence that RNAP, σ70, σ38, Fnr, and (p)ppGpp were influential, suggesting early adaptations. We further show evidence that these responses can be promoter sequence dependent and are partially decoupled from changes in single-cell variability. Moreover, the responses of interacting genes were correlated, suggesting coordination. Later, the transcriptome partially realigned with the control, but 272 genes behaved consistently with long-term adaptation. Several exhibited correlated dynamics that can be explained by interactions, which form three independent, star-like modules of adaptive genes controlled by gcvB, cdaR, and lldR, respectively. Finally, we found that orthologous genes of the evolutionarily distant pathogen Mycobacterium tuberculosis have correlated response strengths to rifampicin, suggesting that our observations may be common across bacterial species. Overall, the results suggest that the initial effects of sublethal rifampicin concentrations on E. coli's genome-wide transcriptional levels are dampened over time and then followed by a distinct state influenced by the gene network that may contribute to adaptation. These findings may assist in developing new strategies to disrupt bacterial adaptation to rifampicin.In natural environments, exposure to sublethal antibiotic (AB) stress is a common phenomenon that enhances the emergence of AB resistance. We dissected the genome-wide transcriptional program of Escherichia coli responsible for its initial response and subsequent adaptation to rifampicin. We show that the transcription factor network plays a major role in this program and provide evidence that the transcriptome response patterns are conserved in the evolutionarily distant pathogen Mycobacterium tuberculosis. Our findings may assist in developing new strategies to disrupt bacterial adaptation to rifampicin.
The establishment of the gut microbiota in early life is fundamental to lifelong health, yet this process remains poorly explored outside Western and high-income settings. We investigated microbial maturation in a non-Western context, by longitudinally characterizing the gut microbiota of 20 healthy infants from urban Bangladesh (n = 984 fecal samples) across the first 2 years of life. Microbiota development followed a three-staged successional pattern characterized by a progressive increase of microbiota diversity, closely resembling patterns described in Western populations. Comparative analysis with a Belgian infant cohort showed broadly conserved maturation dynamics but population-specific differences in core bacteria, including Segatella, which was characteristic of Bangladeshi infants. Segatella's role in the developing gut remains unclear, as high abundances were associated with disease in this cohort. Transient disruptions in maturation also coincided with episodes of illness. The second year of this cohort coincided with the onset of the COVID-19 pandemic where differences in the relative abundance of several key taxa were detected. These insights expand our understanding of healthy infant gut microbiota development across populations and emphasize the need to consider sociocultural and environmental factors, including global disruptions, in shaping early-life microbial ecosystems.The first years of life represent a critical period during which the gut microbiota is established, with lasting implications for long-term health. However, current knowledge of this process is derived largely from studies conducted in Western and high-income populations even though gut microbiota composition is known to vary across geographic locations. In this study, we longitudinally characterized the gut microbiota development in a healthy infant cohort from urban Bangladesh, showing that geographically distinct populations can harbor different microbial communities while following similar developmental patterns. We further observe that illness can temporarily influence microbiota maturation, and that part of the microbiota maturation period in this cohort overlapped with a major societal disruption, the COVID-19 pandemic. Together, these findings illustrate the importance of geographic, cultural, and environmental context when defining healthy gut microbiota trajectories and contribute to a more inclusive, globally representative framework for understanding early-life microbiota development.
Despite recent advances in bacterial profiling across the three anatomical types of pediatric short bowel syndrome (SBS), the gut multi-kingdom remains unexplored. We characterized the four-kingdom gut microbiota using modified DNA extraction and deep shotgun metagenomic sequencing of fecal samples from 26 healthy controls and 34 pediatric SBS patients comprising three types (8 SBS I, 15 SBS II, and 11 SBS III). Overall, children with SBS exhibited a significant reduction in α-diversity compared with controls, with no difference observed among SBS types. Compared with controls, the proportion of archaea was significantly decreased in all SBS types, while bacteria, fungi, and viruses remained similar across types. Different types of SBS exhibited distinct microbial signatures: SBS I was enriched with pathogens (such as species from the Streptococcus and Klebsiella genera); SBS II was marked by a depletion of beneficial short-chain fatty acid-producing species (such as Faecalibacterium prausnitzii); and SBS III displayed loss of bile acid-metabolizing species, alongside a significant expansion of Lactobacillus species. Ecological networks were rewired in SBS, with disruption pronounced in SBS I. Functional analysis revealed that core metabolic pathways were markedly suppressed in SBS I compared with controls. Integrated correlation analyses revealed ileocecal valve loss as the primary determinant of SBS I microbial profiles, linking it to impaired secretory functions and hepatic injury. Our findings deciphered type-specific alterations in the multi-kingdom microbiota and their functional profiles, providing the basis for designing precision microbial therapies aimed at improving long-term outcomes for children with SBS.IMPORTANCEPediatric short bowel syndrome (SBS) is a primary cause of intestinal failure, yet prior research characterizing the gut microbiota has focused almost exclusively on bacteria. In this study, we characterized the multi-kingdom microbiome (including bacteria, fungi, archaea, and viruses) across the three anatomical types of pediatric SBS. We found that different SBS subtypes showed distinct microbial patterns: SBS I was enriched in pathogens, SBS II exhibited a depletion of beneficial short-chain fatty acid-producing species, and SBS III was characterized by a loss of bile acid-metabolizing microbes with an expansion of Lactobacillus. Functional analysis showed that SBS I had markedly suppressed core metabolic pathways, and integrated analyses identified the ileocecal valve as a key determinant of microbial gene profiles, with its loss linked to impaired secretion and liver injury. These findings provide a comprehensive multi-kingdom view of the pediatric SBS microbiome and highlight anatomical determinants shaping host-microbiome dysfunction.
Mycoplasma gallisepticum (MG) is a major pathogen causing chronic respiratory disease in chickens, and the MG-attenuated vaccine (MGAV) often fails to induce effective mucosal immunity. Radix Pseudostellariae saponins (RPS), important bioactive components of the traditional Chinese herb, have been utilized for immunomodulation, although the underlying mechanisms remain to be elucidated. In this study, chickens were randomly assigned to groups receiving intranasal MGAV alone, MGAV plus RPS at different doses, or appropriate controls. Serum, tracheal mucosa, and respiratory lavage samples were collected for antibody measurement, cytokine profiling, histological assessment, and metagenomic/metabolomic analyses. RPS significantly increased MG-specific antibody levels and enhanced expression of the immunomodulatory cytokines, while suppressing that of pro-inflammatory factors. It also promoted tracheal mucosal integrity by elevating goblet cell numbers and mucosal epithelial height. Through metagenomic and metabolomic analysis, RPS was found to enrich the respiratory bacterium Lactobacillus sp. UMNPBX13, which was positively correlated with the metabolite phosphatidylcholine (PC). Intranasal administration of Lactobacillus increased mucin 2 (MUC2) and PC levels in tracheal mucosa. Similarly, PC combined with MGAV enhanced mucosal immunity by modulating cytokines and increasing MG-specific antibodies. These findings demonstrate that RPS enhances MGAV-induced respiratory mucosal immunity and provides a novel adjuvant strategy for improving poultry respiratory disease vaccines by modulating the respiratory microbiota, specifically through enrichment of Lactobacillus sp. UMNPBX13, which promotes PC production.IMPORTANCEMycoplasma gallisepticum (MG) constitutes a notable challenge to poultry health. Although the MG-attenuated vaccine (MGAV) is widely used for disease control, it often fails to induce robust mucosal immunity. Here, we demonstrate that intranasal co-administration of Radix Pseudostellariae saponins (RPS) with MGAV augments mucosal immunoprotection. Integrated metagenomic and metabolomic analyses revealed that RPS enriches respiratory Lactobacillus, which in turn promotes the production of its key metabolite phosphatidylcholine (PC), thereby supporting mucosal immune enhancement. These findings offer a promising strategy for improving poultry vaccine efficacy and may provide a basis for future exploration of mucosal vaccination approaches in other animal species.
This prospective cohort study investigated the longitudinal development of the salivary bacteriome, virome, and metabolome during early infancy. We assessed the associations between oral bacteria, viruses, and metabolites from 10 mother-infant dyads, with oral samples collected at 1 and 2 years of age. Forty saliva and plaque samples underwent untargeted metabolomic analysis, and infant saliva samples underwent metagenomic sequencing. Maternal salivary and plaque metabolomic profiles remained largely stable, whereas infant profiles were clearly separated from maternal profiles and changed with age. Notably, infant dental plaque metabolism underwent more substantial changes from year 1 to year 2 than saliva, with age-dependent metabolite shifts mainly involving energy, amino acid, nucleotide, and lipid metabolic pathways. Our findings also revealed significant developmental shifts in salivary bacteriome, virome, and functional pathway profiles during early childhood. The most abundant oral bacteria in early life, comprising over 75% of total abundance, included Veillonella, Streptococcus, Rothia, Prevotella, Neisseria, and Actinomyces species. While human viruses like Roseolovirus were detected, bacteriophages constituted the majority of the virome. Comparing infants at year 1 and year 2, we identified differentially abundant bacteria, viruses, metabolic functional pathways, and specific metabolites. We observed associations between bacteria and viruses, noting that these cross-kingdom relationships attenuated as infants grew. The study results underscore the complex and dynamic development of the oral microbiome, virome, and metabolome during early childhood.IMPORTANCEThe human oral cavity undergoes substantial microbial and metabolic development during early childhood, yet the temporal changes in the infant oral ecosystem remain incompletely understood. In this study, we longitudinally profiled the salivary metabolome, bacteriome, and virome of infants at 1 and 2 years of age. We demonstrated that the infant oral metabolome undergoes substantial developmental shifts, particularly in pathways related to energy, amino acid, and lipid metabolism; whereas maternal metabolic profiles remained stable over the same period. Furthermore, our results revealed the dynamic assembly of infant salivary virome and bacteriome and their associations with the functional pathways and metabolites. These findings provide new insights into the complex and dynamic development of the oral microbiome, virome, and metabolome in early infancy.
Physical inactivity contributes substantially to global disease burden, yet the physiological mechanisms underlying exercise motivation remain poorly understood. The gut-brain axis presents a potentially modifiable target for behavioral intervention. Emerging evidence demonstrates that the gut microbiota influences motivated behaviors, but the specific metabolic functions and physiological mechanisms mediating these effects remain poorly defined. Here, we demonstrate that the predicted fermentation capacity of the gut microbiota influences voluntary wheel running (VWR) acquisition and neuroendocrine integration during exercise in C57BL/6J mice. Antibiotic-induced microbiome depletion reduced VWR acquisition, while shifting predicted function toward aerobic respiration and away from anaerobic fermentation. Supplementation with short-chain fatty acids, the primary fermentative products, restored normal VWR activity in microbiome-depleted mice. Conversely, 4-week dietary pretreatment with 2.5% prebiotic fiber (inulin) increased predicted fermentative capacity of the microbiota and VWR activity above baseline levels. Microbiome manipulation produced bidirectional dysregulation of corticosterone responses to exercise: acute antibiotic depletion increased post-exercise concentrations, while germ-free development decreased them, despite elevated striatal catecholamines. This exercise-specific uncoupling reveals microbiome-dependent integration of metabolic demand signals in the coordination of sympathetic and hypothalamic-pituitary-adrenal axis responses. Furthermore, the inulin-induced enhancement in VWR activity was associated with increased striatal histamine concentrations following exercise, suggesting additional mechanisms of neuromodulation. These findings demonstrate that the fermentative capacity of the gut microbiota influences exercise motivation and neuroendocrine regulation, providing novel insights into dietary interventions targeting physical activity. IMPORTANCE:Physical inactivity is a leading cause of global morbidity and mortality, and our lack of understanding of the biological forces driving motivation to exercise limits our ability to develop interventions that enhance engagement. Using a rodent model of voluntary exercise along with microbiota depletion and metabolite replacement, we uncovered that the gut microbiota and its capacity to ferment dietary components into short-chain fatty acids drive exercise habit acquisition and help facilitate coordination between neurochemical signals and systemic stress hormones during exercise. Additionally, microbiome depletion "uncoupled" these systems, resulting in dysregulated stress responses during forced exercise. Finally, we showed that enhancement of microbiota fermentation capacity via dietary addition of prebiotic fiber was able to increase exercise engagement while also enhancing concentrations of histamine, a neuromodulator that potentiates locomotor activity, in the striatum. These findings suggest that the gut microbiome is a modifiable target for behavior change that facilitates integration of metabolic demand in neuroendocrine activity. Collectively, this work provides a mechanistic foundation to support the use of dietary interventions in sedentary populations to start exercise habits.
As ecologists increasingly use metagenomic time series to track evolution in the wild, there is a risk of misinterpreting ecological dynamics as rapid adaptation. This Perspective identifies methodological limitations that generate misleading signatures of microbial evolution. A primary issue is confusing evolutionary change (driven by de novo mutation or horizontal gene transfer) with ecological lineage turnover, such as seasonal oscillations or the reactivation of dormant lineages. Current metagenome-assembled genomes can collapse micro-diverse lineages and decouple adaptive mobile elements, creating inaccurate genomic signatures of sweeps or stasis. To address these issues, I propose a framework integrating long-read sequencing, pangenome graph theory, and forward-time simulations to model populations as temporal genetic networks and better resolve microbial evolutionary dynamics.
Viruses are increasingly recognized as important players in soil ecosystems, but the active lytic virus populations that influence microbe-mediated terrestrial ecosystem processes remain mostly uncharacterized. Here, we trace 13C-labeled glucose from host microorganisms into virus genomic DNA to identify virus populations actively involved in soil carbon (C) cycling, i.e., viruses that lysed 13C-incorporating microbes. We present experimental evidence of isotope labeling (i.e., lytic activity) of more than 5,000 virus populations. The active viruses lysed hosts from 197 microbial families across 28 prokaryotic phyla. Viral lysis was greater in C-limited agricultural soils compared with C-rich forest soils, highlighting C availability/inputs as key factors that mediate virus life cycles. Active viruses disproportionately lysed microorganisms in the Bacillota, Bacteroidota, and Pseudomonadota phyla, likely reflecting glucose-induced growth responses of microbial copiotrophs within those groups. Supporting this, we observed that the degree of virus genome isotope labeling was positively correlated with the growth potential of the microbial hosts. Furthermore, the active viruses exhibited unique genomic characteristics compared to the inactive viruses, including a greater prevalence of lysogeny-associated genes and distinct profiles of putative auxiliary metabolism genes in the active virus genomes. Overall, our results demonstrate a link between microbial growth traits and virus activity and suggest that substrate-induced viral lysis significantly influences microbial population turnover in soil. Our results also show that virus activity in response to C inputs is highly variable among soil contexts, with implications for the varying ecosystem-scale influences of viruses among terrestrial environments.IMPORTANCEIn this study, we used a new method of detecting active viruses that lyse (kill) their microorganism hosts in soil. We accomplished this by tracing "heavy" versions of carbon from soil microorganisms into the DNA of their virus parasites. This showed that there are thousands of different potentially active virus populations in soil and that the microorganisms that get lysed the most are those that grow the fastest. However, the activity of viruses was very high in some ecosystems (agricultural soils) and much lower in others (forest soils). Our results show that viruses can play an important role in influencing soil microbial communities, but that the size of their effects is very different in different types of ecosystems. Our study also describes a new method that can be extended to further explore the roles and importance of viruses across different environmental conditions and different ecosystem types.
The accelerating deposition of RNAseq data over the past decade has motivated the development of advanced transcriptomic data analytics that can operate on a large number of samples. One successful approach is to apply independent component analysis (ICA) to large prokaryotic transcriptomic compendia to decompose them into independently modulated gene sets, called iModulons. Here, we review the data science principles underlying ICA-based transcriptome decomposition, computational workflows that support its routine application, and iModulonDB infrastructure that hosts and disseminates the resulting decompositions. We present iModulonDB 3.0 that contains 53 species and 71 ICA decompositions across 33,062 RNA-seq samples, with several well-sampled species (e.g., Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa) represented by more than one compendium. With 71 standardized decompositions, we demonstrate systematic cross-species comparison of species-specific iModulon structures. This comparison identifies a shared "regulatory toolkit" of 13 modules conserved across distantly related bacteria, alongside a long tail of lineage-specific programs. We assess the design principles and limitations governing iModulon reconstruction and computation. Together, these advances position the iModulon framework as an accessible, community-driven approach for reading accumulating public transcriptomes as reusable regulatory programs, enabling biological discovery and module-level design in synthetic biology.
Owing to its compositional and chemical complexity, much of the gut microbiota metabolome remains poorly characterized. Aromatic amino acids (AAAs) have a history of being privileged substrates for the biosynthesis of diverse bioactive metabolites and thus represent a potentially rich source of bioactive molecules within the microbiota metabolome. In this study, we leveraged 13C- and 2H-labeled aromatic amino acids and untargeted liquid chromatography-mass spectrometry (LC-MS) to profile AAA-derived metabolites produced by 80 phylogenetically diverse human gut bacterial isolates. Collectively, we found 93 unique LC-MS features, majority of which, predominantly produced by Clostridioides difficile, were identified as N-acyl amino acids. C. difficile produced the highest levels of the AAA-derived phenylacetic acid and phenylpropionic acid, exceeding all Bacteroidetes and Proteobacteria strains in our panel. C. difficile's uniquely diverse N-acyl amino acids have the potential to serve as biomarkers for C. difficile colonization and mediators of C. difficile-specific host interaction.IMPORTANCEThe bacterial metabolome is a key component of the microbiota's effect on host physiology, but identifying small molecules that potentially drive this interaction has remained a challenge. This study uses high-throughput and quantitative mass spectrometry metabolomics to show that Clostridioides difficile uniquely converts amino acids into at least 28 N-acyl amino acids, a metabolite family historically linked to diverse bioactivities. Additionally, in C. difficile cultures, high levels of phenylacetic acid, the precursor of 6 N-acyl amino acids, are of interest because previous studies have mechanistically linked microbially produced phenylacetic acid to cardiovascular disease via β2-adrenergic receptor (β2AR) signaling. The identification of species-specific metabolites produced by commensal bacteria provides not only compounds that could serve as sensitive biomarkers of colonization but also helps support the formulation of mechanistic hypotheses regarding how individual species influence their host.
The biosynthetic capacity of a cell governs the production and exchange of amino acids. Given the distinct metabolic origins and intracellular requirements for amino acids, it is essential to establish quantitative amounts of internal amino acid pools and how these change across growth phases. Using Saccharomyces cerevisiae, here we establish an absolute, quantitative blueprint of the intracellular and extracellular amino acid economy, defining the fluxes of production, secretion, and consumption across 24 hours of cell growth. While the intracellular pool is dominated by a group of amino acids, their relative proportions continuously change over time. The extracellular pool is notably distinct in terms of composition and amounts. Only select amino acids are public goods secreted in significant amounts, and a subset of these (Ala, Val, Gln, Trp, and Phe) are subsequently re-consumed. Five amino acids, Asp, Lys, His, Arg, and Met, remain "privatized" even in nutrient abundance. We demonstrate that the strictly privatized amino acid Asp continuously sustains diverse carbon metabolism, while the public Ala is utilized following carbon depletion. Furthermore, nitrogen limitation triggers a significant shift toward storage, privatizing otherwise abundant nitrogen-rich compounds like Gln, Asn, and Pro. We thereby rationally establish pairs of stable synthetic communities of paired public good auxotrophs that show effective growth. Our results identify frameworks for feasible amino acid trade and provide a basis for engineering stable, synthetic communities of amino acid auxotrophs.IMPORTANCEAmino acids are central to a metabolic economy and are extensively exchanged between cells; yet, the scale of this economy remains unknown even in model microbes. This study establishes a quantitative blueprint of the amino acid economy in Saccharomyces cerevisiae by mapping production, secretion, and consumption fluxes. The findings reveal a distinction between public goods-such as alanine, which is secreted and re-consumed-and privatized resources, such as aspartate, which cells retain to sustain carbon metabolism. These pools shift across growth phases and move toward privatization during nitrogen limitation. By defining these frameworks, this study enables the rational design of stable, synthetic communities of auxotrophs.
Polymyxins are often last-resort antibiotics against high-priority Gram-negative pathogens, particularly Acinetobacter baumannii. However, most mechanistic studies to date have overlooked the heterogeneity within bacterial populations during polymyxin treatment. Using time-lapse imaging and propidium iodide (PI) staining, we observed that a subset of PI-positive (PI+) cells, which are traditionally considered non-viable, were capable of regrowth. This unexpected scenario promoted further investigation into the distinct metabolic responses of PI+ or PI-negative (PI-) subpopulations following polymyxin exposure. By combining a synthetic fluorescent polymyxin probe, FADDI-043, with fluorescence-activated cell sorting (FACS), we isolated PI+ and PI- cells and profiled their metabolic responses. PI+ cells exhibited increased levels of phosphatidylethanolamine, likely to compensate for the severe membrane damage by polymyxins. In contrast, PI- cells, where polymyxin interacted with bacterial membranes without causing extensive damage, demonstrated broader metabolic adaptations. Notably, arginine metabolism was uniquely upregulated in the PI- group, and exogenous arginine supplementation conferred protection against polymyxin treatment. Collectively, this is the first study to demonstrate subpopulation-specific metabolic responses to polymyxins, highlighting dynamic and heterogeneous bacterial adaptations. Our findings underscore the importance of single-cell analysis to unravel antibiotic mechanisms and may inform novel metabolic reprogramming strategies to enhance antibiotic efficacy and minimize resistance emergence.IMPORTANCEMultidrug-resistant Acinetobacter baumannii is designated a World Health Organization "critical priority" pathogen, and polymyxins remain the few effective treatment options, particularly in low- and middle-income countries. However, polymyxin heteroresistance poses a major global clinical challenge, and its mechanistic basis remains poorly defined. Most antimicrobial studies rely on population-level measurements, obscuring the biological consequences of phenotypic heterogeneity. Here, we demonstrate that a subset of polymyxin-treated, propidium iodide-positive (PI+) A. baumannii cells, typically classified as non-viable, retain the capacity to regrow. By isolating PI+ and PI-negative (PI-) subpopulations, we uncover distinct metabolic adaptations under polymyxin exposure: PI+ cells exhibit elevated phosphatidylethanolamine levels, whereas the PI- cells activate their arginine metabolism. These findings reveal an unrecognized layer of metabolic heterogeneity underlying polymyxin exposure. Our work challenges conventional interpretations of viability assays and highlights the importance of subpopulation-resolved analyses for identifying metabolic vulnerabilities to enhance polymyxin efficacy while limiting resistance emergence.
The human lung microbiome is increasingly recognized as a key player in drug metabolism, yet it remains largely understudied. To replicate this complex physiological environment in a controlled setting, we developed a simplified artificial lung microbiome model composed of four representative bacterial species: Pseudomonas koreensis, Rothia aeria, Neisseria cinerea, and Streptococcus downei. We successfully established a stable 10-day co-culture at 34°C using brain heart infusion medium, as validated by quantitative PCR, viability PCR, and conventional microbiological methodologies. Integrated bioinformatic analyses revealed a variety of potential microbe-microbe interactions, which were supported by metaproteomic analysis using mass spectrometry. Our model provides a foundation for in-depth studies, such as, for example, the effects of pulmonary drugs on the lung microbiome, and how, in turn, the microbiome may influence therapeutic outcomes.IMPORTANCEOnce thought to be sterile, the lung microbiome is now understood to host a dynamic microbiome capable of influencing respiratory health and disease. Understanding interactions among the microbes within this community is essential, as these relationships may drive disease progression or foster resilience in both acute and chronic inflammatory conditions. We developed a reproducible lung microbiome model comprising Pseudomonas koreensis, Rothia aeria, Streptococcus downei, and Neisseria cinerea. Simplified models enable controlled studies to dissect specific microbial interactions, laying the foundation for insights into lung microbial ecology. In the future, more complex models will enhance our understanding of microbial roles in disease outcomes, with our platform serving as a basis for testing therapeutic strategies.
Plastic biodegradation in natural environments is increasingly recognized as a multi-organism process; however, the mechanisms enabling coordinated depolymerization and metabolism of polyethylene terephthalate (PET) remain poorly understood. Previously, we demonstrated that a full consortium containing three Pseudomonas and two Bacillus strains isolated from hydrocarbon-rich coastal soils of Galveston Bay, Texas, can synergistically depolymerize PET plastic and utilize it as a sole carbon source, a capacity not observed in individual isolates. In this report, using integrated comparative genomics, proteomics, and chemical analyses, we show that PET degradation in this system reflects exaptation of hydrocarbon metabolism, reinforced by metabolic division of labor. Within this naturally occurring consortium, Bacillus strains persist under environmental stress, establish biofilms, and perform essential secondary hydrolysis, while Pseudomonas strains catabolize aromatic monomers and buffer oxidative stress. Genes supporting these functions are enriched within the accessory genomes of the consortium strains, indicating consortium-enriched horizontal gene transfer. In addition to the canonical two-step hydrolytic pathway well documented in PET biodegradation, we identify a secondary methylation- and redox-associated process, mechanisms where the full consortium acts on the oligomer mono(2-hydroxyethyl) terephthalate (MHET), yielding nearly complete conversion to terephthalic acid and methylated MHET. Together, these findings support a model in which PET degradation is driven by pre-existing hydrocarbon metabolic traits distributed across the native consortium, particularly stress-response and redox pathways, aromatic catabolism, and alcohol catabolism. IMPORTANCE:Environmental plastic degradation is rarely accomplished by a single organism; however, the microbial mechanisms enabling community-level polyethylene terephthalate (PET) plastic breakdown remain poorly understood. This study shows that a bacterial consortium isolated from crude petroleum-contaminated coastal soils degrades PET by coordinating older hydrocarbon, aromatic, stress-response, and redox-associated metabolisms rather than by acquiring a dedicated PET pathway. Predicted horizontal gene transfer events were linked mainly to survival, biofilm formation, and metabolic flexibility, not PET depolymerization itself. These findings shift the focus from searching for single PET-degrading organisms toward understanding how microbial communities combine pre-existing metabolic tools to process synthetic polymers and manage the chemical stress created during biodegradation.
Microbes in natural environments often encounter diverse mixtures of organic compounds, yet how mixed substrate environments and their molecular composition shape microbial phenotypes remains understudied. Here, we examined how a defined fungal exudate mimic (FEM) mixture influences growth and metabolism in Pseudomonas putida KT2440 compared to individual substrates matched for total carbon and nitrogen. Growth on FEM initiated 2 h earlier than growth on glucose alone and exhibited both the lowest lag and shortest time to maximum biomass compared to individual substrates. Fructose was the only individual substrate that supported significantly higher maximum biomass than FEM, but exhibited a nearly 15-fold longer lag phase. Gas chromatography mass spectrometry analysis revealed dynamic temporal patterns of substrate utilization within the FEM mixture, with early preferential utilization of malate, followed by overlapping utilization of multiple substrates between 3 and 8 h. By integrating growth and substrate uptake kinetics with genome-scale metabolic modeling and validating model-predicted pathway activity using temporal proteomics, we show that experimentally constrained model predictions accurately captured substrate utilization dynamics across multiple FEM concentrations, and predicted temporal shifts in the dominant substrates supporting growth. Through this integrative experimental-modeling approach, we demonstrate that the mixed substrate FEM environment elicits an emergent growth phenotype characterized by the lowest lag, shortest time to maximum biomass, and relatively high maximum biomass in Pseudomonas putida, a combination of traits not simultaneously reproduced by any individual substrate. IMPORTANCE:How mixed-nutrient substrate environments influence bacterial growth and metabolism is not well understood and is challenging to predictively model. Using Pseudomonas putida KT2440, we show that growth on a defined mixture of substrates inspired by mycorrhizal fungal hyphal exudates produces a distinct and emergent growth phenotype characterized by a lower lag, shorter time to maximum biomass, and relatively high biomass accumulation when compared to individual substrates alone. This combination of growth traits is not simultaneously reproduced by any individual substrate, even when total carbon and nitrogen are matched. By integrating growth measurements and temporal substrate uptake data with dynamic flux balance analysis and proteomics, we demonstrate that metabolic responses to mixed substrates can be quantitatively interpreted. These findings highlight the importance of studying microbes under chemically realistic conditions and suggest that learning from naturally occurring molecular environments may provide new strategies for engineering microbial growth conditions and improving bioprocess performance.
ABSTRACT Preventing Listeria monocytogenes persistence in food processing environments is challenged by tolerance to quaternary ammonium compound sanitizers such as benzalkonium chloride (BAC). To define the genetic and molecular routes to BAC tolerance and antibiotic cross-resistance, we performed an evolve-and-resequence experiment on three L. monocytogenes strains (three replicate lineages each) passaged under sublethal BAC for ~400–500 generations. All nine BAC-exposed lineages increased BAC minimum inhibitory concentrations (MICs), and several showed elevated tolerance to a commercial sanitizer. Notably, multiple lineages displayed cross-resistance to fluoroquinolones. Whole-genome sequencing of end-point lineages revealed recurrent, parallel mutations in the fepR transcriptional regulator (often involving nonsense mutations), accompanied by missense mutations in the adjacent efflux pump fepA , implicating derepressed multidrug and toxic compound extrusion (MATE) efflux activity in BAC, and fluoroquinolone tolerance. RNA-seq under BAC stress showed consistent upregulation of phosphotransferase system (PTS) genes across evolved lineages. Complementing this, we observed convergent missense mutations in ptsH (encoding the HPr phosphocarrier), including substitutions in the HPr serine phosphorylation region. Biophysical modeling predicted that these HPr variants are strongly destabilizing, consistent with altered PTS signaling under BAC sthe presence of BAC stress tress. Efflux inhibition with reserpine reduced BAC tolerance in fepR / fepA -mutant backgrounds, whereas the effects were minimal in a bcrABC-positive strain (where bcrABC encodes a plasmid-borne BAC resistance efflux cassette), underscoring strain-dependent mechanisms. Together, our data reveal efflux-driven and PTS-linked adaptation to BAC with implications for the emergence of antibiotic cross-resistance and persistence in food production environments. IMPORTANCE Listeria monocytogenes persistence in food facilities poses major public health and economic burdens. We modeled long-term exposure to sublethal benzalkonium chloride (BAC) to approximate conditions similar to those in food processing facility drains and uncovered two complementary routes to sanitizer tolerance: (i) repeated inactivation of the fepR regulator with accompanying alterations in the fepA efflux pump, driving BAC tolerance and fluoroquinolone cross-resistance, and (ii) convergence on mutations in ptsH (HPr) within the phosphotransferase system, supported by RNA-seq upregulation of phosphotransferase system (PTS) pathways and biophysical evidence of destabilized HPr variants. Efflux inhibition reduced BAC tolerance where fepR/fepA was mutated but had limited impact in a bcrABC-positive background (where bcrABC encodes a plasmid-borne BAC resistance efflux cassette), highlighting the role of genetic context. These findings connect sanitizer adaptation to antibiotic resistance and identify PTS signaling as a systems-level contributor, informing surveillance and sanitation strategies in food production.
