Cell Host & Microbe

Plants are colonized by a diverse microbiome, with microorganisms residing inside and outside of plant tissues. Plants can harness the protective traits of their microbial inhabitants to ward off insect pests and fungal pathogens. However, current understanding of the role of commensal interactions on activating the desired microbial genomic traits remains limited. Here we show that biosynthesis of the antifungal 2,5-dihydro-L-phenylalanine (DHP) by the endophytic Streptomyces sp. PG2 is strongly induced upon colonization of Arabidopsis thaliana. DHP production protects the plant from infection by the fungal root pathogen Rhizoctonia solani, both in vitro and in vivo.. We identified the DHP biosynthetic gene cluster (BGC) and showed that heterologous expression of the BGC in the DHP non-producer Streptomyces coelicolor also conferred plant-inducible DHP production. The BGC was also found in plant-associated Gram-negative bacteria, and in Pseudomonas syringae FF5 we again observed strongly enhanced DHP production upon plant colonization. An ecology-inspired elicitor screen showed that L-valine and brassinosteroid hormones elicit DHP biosynthesis in the plant-beneficial Streptomyces sp. PG2, while L-valine also elicited DHP biosynthesis in S. coelicolor. In vivo experiments confirmed the stimulation of antifungal activity in Streptomyces sp. PG2 by L-valine, while brassinolide mutant plants showed reduced DHP induction. Conversely, neither L-valine nor brassinolide elicited the expression of the DHP BGC in the pathogenic P. syringae, revealing important divergence in the responses to plant signaling, which may reflect selectivity in how endosymbionts and pathogens respond to host cues. Collectively, our data demonstrate that plant colonization can elicit the biosynthetic potential of root-associated microbes, thereby enhancing plant resilience.

Antibiotic tolerance paves the way for acquired resistance in bacterial pathogens. However, the mechanisms of tolerance and its evolutionary role in acquired resistance in pathogenic fungi, particularly molds, remains elusive. Here, we identified an Inhibitor of Growth domain protein (IngB) as a novel epigenetic regulator of azole tolerance in Aspergillus fumigatus. The loss of ingB promotes supra-MIC growth on agar surface despite susceptible MICs in standardized assays. Moreover, established {Delta}ingB biofilms are less susceptible to azoles in vitro and in vivo. Subsequent exposure of the tolerant strain to high azole concentrations resulted in rapid acquired resistance, most notably a frameshift mutation in a putative 20S proteasome maturation protein, UmpA, while the susceptible wildtype strain failed to acquire adaptive mutations. The data suggest that IngB-mediated tolerance provides an epistatic background for the emergence of azole resistance. Our work shows drug tolerance facilitates resistance emergence in a critical fungal pathogen. ImportanceWhile antimicrobial drug resistance causes a significant adverse effect on human health, drug tolerance can also lead to insufficient pathogen clearance, resulting in infection relapse. However, the mechanisms of antifungal drug tolerance and its evolutionary role in acquired drug resistance in pathogenic fungi, particularly the molds, remains elusive. We identified IngB as a novel regulator of azole tolerance in Aspergillus fumigatus. Importantly, loss of IngB leads to rapid azole drug resistance under azole-selective pressure. Our work identifies a novel regulator of antifungal tolerance and suggests antifungal drug tolerance can pave the way for resistance emergence in a critical fungal pathogen.

The ocular surface is a mucosal tissue that is constantly exposed to environmental antigens and potential pathogens. Human microbiomes play a critical role in the balance of surveillance and inflammation at sites of colonization. Historically, the investigation of the ocular microbiome has been difficult due to its paucibacterial nature and the inhospitable environment of the ocular surface. Despite this, Corynebacterium mastitidis (C. mast) developed a unique ability to colonize the eye and elicit a protective immune response characterized by induction of IL-17 from {gamma}{delta} T cells and protection from corneal infection. Therefore, we sought to understand the unique bacterial machinery that C. mast utilizes to colonize the eye and how it affects the induction of an eye-specific immune signature. Using a C. mast transposon mutant library, we identified a mutant that completely lacked an ability to form biofilm, colonize the eye, and induce in vivo immunity. Whole genome sequencing revealed a disruption in the sortase F gene, which anchors proteins to the cell wall of C. mast, governing biofilm formation and tethering of adhesins to the cell surface. Additionally, we show that mutation in individual C. mast adhesins does not affect ocular colonization or immune induction. By understanding the molecular mechanism of ocular microbial colonization, this work advances our understanding of how bacteria colonize and induce immune responses on the eye, providing a foundation for developing novel therapeutic strategies against ocular infections.

Plant-associated microbial communities play a critical role in plant health and disease resistance, but the mechanisms which reshape these communities during pathogen infection are poorly understood. In this study, we investigated how infection of maize by the smut fungus Ustilago maydis is functionally linked with the bacterial phyllosphere microbiome and explored the role of an antimicrobial effector GH25 in fungal infection. Using a combination of culture-dependent and culture-independent approaches, we compared the leaf microbiomes of infected and uninfected plants. We observed a significant increase in microbial abundance and pronounced shifts in community composition and identified distinct health-associated (HCom) and disease-associated (DCom) bacterial communities. To assess whether U. maydis directly manipulates the microbiome, we tested the antimicrobial activity of the antimicrobial effector GH25 against isolated strains. Notably, all HCom bacteria were sensitive to GH25 and co-inoculation of HCom bacteria with a U. maydis {Delta}gh25 knockout mutant significantly reduced fungal virulence. In contrast, DCom exhibited minimal sensitivity to U. maydis and did not affect the virulence of U. maydis {Delta}gh25. Functional profiling revealed infection-associated shifts in predicted metabolic potential, consistent with U. maydis induced leaf tumors being strong sink tissues. Together, the data shows that U. maydis infection reshapes the maize phyllosphere microbiome through a combination of effector-mediated antimicrobial activity and host metabolic reprogramming.

Neutrophils and monocytes are the main fungal effector cells in restricting Blastomyces dermatitidis (Bd) and other fungi at the respiratory mucosa. However, understanding how phagocytes become activated and recruited to the site of infection is still incompletely understood. Innate lymphocytes and myeloid cells have been found to communicate and play an essential part in activating neutrophils and other effector cells to kill fungi. Here, we identified that Signaling Lymphocytic Activation Molecule 1 (SLAMF1) is a key host immune receptor involved in orchestrating a cellular and molecular signaling network that leads to the activation of phagocytes. By using mice to conditionally eliminate SLAMF1 receptor expression on innate CD4+ or TCR{gamma}{delta}+ T cells, we uncovered that these innate lymphocytes augment neutrophil killing of Bd in a SLAMF1 dependent manner. SLAMF1 expression on neutrophils enabled homotypic SLAMF1:SLAMF1 interactions with innate CD4+ T cells, which prompted release of soluble factors that activated neutrophils to kill fungi. Our work furnishes new mechanistic insight about the role of SLAMF1 in mobilizing innate immune cells to induce phagocyte-driven killing of inhaled fungi. Author SummaryEmerging fungal diseases represent a significant and growing global public health threat fueled by increased anti-fungal resistance and rising number of immunocompromised individuals. Most fungal infections are respiratory and occur when inhaled fungal spores settle in the lungs and cause inflammation or tissue damage. The innate immune system is the first line of defense in the lungs but the mechanisms by which the host immune system becomes activated and mounts a protective response is still not completely understood. We identified a receptor on innate immune cells that facilitates communication between cells and recruits and activates killer cells that engulf and destroy the fungal pathogen. We uncovered that the receptor on the cell surface of innate immune cells mediates its function through cell contact and induction of soluble factors. Our work offers new mechanistic insight about how the innate immune system becomes activated by the presence of fungi and orchestrates an effective host response. We envision that soluble receptor could be harnessed for future anti-fungal therapy.

Secreted mucins are the major component of the mucus layer that protects intestinal epithelial surfaces by blocking excessive interactions with the microbiota. Mucins are complex glycoproteins decorated with over 100 different O-glycans. Some bacteria can utilize mucins and excessive degradation has been associated with disruption of the mucus barrier and inflammation. Despite the importance of mucins, a detailed enzymatic pathway by which gut bacteria degrade colonic mucin O-glycans and the impact of this process on gut colonization are unknown. Here, we identified >100 genes that are expressed by the symbiont Bacteroides thetaiotaomicron during growth on different O-glycan substrates, revealing effects of glycan structure on gene expression. The characterization of 33 glycoside hydrolase enzymes revealed the pathway for colonic O-glycan degradation by this bacterium. In vivo competition experiments show that multiple exo-acting enzymes targeting mucin capping structures are central to gut colonization and may provide targets to inhibit bacterial mucin degradation.

Early colonisation by bifidobacteria is crucial for infant health, with Bifidobacterium longum subspecies (BL.) dominating the early gut microbiome. However, the interactions between these bacteria and their viruses remain poorly characterised. Here, we applied genomics-based approaches to examine BL. prophage composition and dynamics in infants, as well as their antagonistic and mutualistic evolutionary strategies. Across 213 metagenome-assembled genomes recovered from 139 infant faecal samples in the Dutch Lifelines NEXT cohort, 286 previously undescribed prophages were identified and analysed. Comparative genomics revealed extensive viral diversity, evidence of historical recombination, and widespread counter-defence, with [~]80% of prophages encoding anti-CRISPR or CRISPR-evasion proteins. Approximately half of prophages encoded metabolism altering genes. Notably, prophages and host CRISPR spacer arrays were highly stable across longitudinal samples, indicating stable phage-host associations during early life. Together, these findings show that BL. prophages employ antagonistic and synergistic strategies to maintain infectivity and long-term persistence in the infant gut.

Thoeris immune systems protect bacteria against invading phages through production of a signaling molecule by ThsB that activates the effector ThsA. Across the four defined types, the two most dominant (I and II) have been associated with abortive infection, with type I acting through the depletion of the essential coenzyme NAD+. Here, we show that a previously uncharacterized type II Thoeris system from Escherichia coli ATCC 25922 deviates from this paradigm. This system consists of the effector protein ThsA and two distinct signaling proteins ThsB1 and ThsB2. Heterologous expression of thsA and thsB2 confers anti-phage defense, while thsB1 is cytotoxic when expressed in common E. coli lab strains. Furthermore, while phage infection drives growth arrest, we could not detect any measurable decrease in NAD+ levels as well as standard markers of cell death. Together, these results suggest that Thoeris contains even greater functional diversity within the defined system types.

The Streptococcus pneumoniae capsule is a major determinant of virulence, yet whether bacteria actively remodel it during infection remains unclear. Studying Swiss and South African clinical isolates (serotypes 1, 6B, 8, 12F, 19F, and 35B), we identified a rapid, tissue-specific response: capsule thickness increased within hours upon co-exposure to host cells and tissues. Only two 12F strains failed to thicken. Thickening was greatest in brain tissue, moderate in serum, and absent on the epithelium. This adaptation occurred independently of cod locus phase variation and nutritional factors, and was instead driven by a soluble, thermostable host signal (<3 kDa). Thickening correlated with neuroinflammation but did not require it, as it also occurred in contact with resting brain immune cells. It exacerbated meningitis in mice and enhanced bacteremia. Once induced, capsule thickening dampened inflammatory responses, coinciding with downregulation of pneumolysin, a major pro-inflammatory toxin. Genetic analysis of the non-thickening 12F isolates, together with targeted mutagenesis, identified two independent determinants of capsule-thickness modulation: a specific promoter-proximal element and SPD_0642, a conserved putative transporter encoded outside the capsule operon. Both contributed to the host-induced thickening phenotype. Pneumococci therefore rapidly remodel their surface in response to tissue-specific cues within the host, in a manner distinct from stochastic phase variation outside it. ImportanceMany bacteria are covered by a slimy outer layer, known as a capsule, that helps them evade the immune system. The amount of this layer can influence how easily harmful bacteria cause disease. Until now, scientists knew that bacteria can turn capsule production on or off through changes in their DNA. In this study, we show that Streptococcus pneumoniae, a common cause of serious infections, can also adjust its capsule in another way. It senses soluble signals from the tissues it enters, allowing it to recognize where it is in the body and to gradually change the thickness of its protective outer layer. This finding offers a new way of understanding how bacterial infections develop and may point to new treatment strategies.

Defoliating (D) strains of the vascular wilt fungus Verticillium dahliae cause severe yield losses in cotton and olive worldwide, yet the genetic basis underlying this pathotype has remained unknown. Here, we combined comparative genomics, functional genetics, structural analysis, and phylogenomics to uncover the molecular determinant of defoliation. We identified a small, D pathotype-specific genomic region encoding two duplicated secreted effector genes. Simultaneous deletion of both gene copies abolished pathogenicity and defoliation in cotton, olive, Nicotiana benthamiana, and Arabidopsis thaliana, whereas single deletions reduced virulence and genetic complementation restored disease symptoms. Conversely, expression of the D effector in non-defoliating strains was sufficient to induce defoliation. Moreover, or exogenous application of the purified protein, induced wilting and defoliation as well. Structural analyses revealed that D homologs share a conserved but previously uncharacterized protein fold and are distributed across Verticillium and Fusarium species, exhibiting functional diversification and host-specific activity. Phylogenomic and genomic context analyses indicate repeated horizontal transfer events mediated by giant transposable elements known as Starships. Together, our findings identify the D effector as a central pathogenicity factor that drives defoliation and virulence, and demonstrate how Starship-mediated horizontal gene transfer shapes the emergence and dissemination of an agriculturally devastating fungal trait.

Dysbiosis and bacterial pathobionts contribute to inflammation in IBD and CRC, yet the molecular drivers of this process remain unclear. We identify the Klebsiella pneumoniae type VI secretion system (T6SS) as a key promoter of intestinal inflammation and tumor progression. Metagenomic analyses revealed enrichment of T6SS encoding genes in the gut microbiota of IBD patients during inflammatory flares. In zebrafish and mouse models, K. pneumoniae T6SS activity exacerbated inflammation and promoted colorectal tumor growth. Mechanistically, T6SS firing enhanced the secretion of LPS via outer membrane vesicles (OMVs), driving NF-{kappa}B activation and interferon signalling in host cells. In vivo, T6SS-dependent inflammation was associated with the expansion of regulatory T-cell subsets and an immunosuppressive tumor microenvironment. These findings redefine the T6SS as a microbial determinant of host inflammation and cancer progression, highlighting T6SS inhibition as a potential therapeutic approach for IBD and CRC. HighlightsO_LIT6SS-encoding Enterobacteria are enriched in the gut microbiota of IBD patients C_LIO_LIKlebsiella pneumoniae T6SS exacerbates colitis in mice C_LIO_LIT6SS activity enhances outer membrane vesicle secretion and LPS release C_LIO_LIT6SS promotes colorectal tumorigenesis and immune dysregulation C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/715367v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1c99341org.highwire.dtl.DTLVardef@e2d0eborg.highwire.dtl.DTLVardef@1021387org.highwire.dtl.DTLVardef@1502c97_HPS_FORMAT_FIGEXP M_FIG C_FIG

The gut microbiome composition varies across human populations, but its characteristics and determinants in a multi-ethnic setting remain incompletely understood. Singapore, a multicultural city-state, provides a context in which culturally diverse groups share a broadly similar built environment. Using the Health for Life in Singapore (HELIOS) cohort, we profiled the gut microbiome of ethnic Chinese, Indian, and Malay participants (n=861) who resided in the country. Despite substantial overlap in the overall microbial compositions, each group displayed distinct microbial signatures that paralleled culturally rooted dietary habits. Specifically, ethnic Indian participants showed enrichment of multiple Bifidobacterium species associated with greater intake of traditional grain-based staples such as idli and thosai; ethnic Malay participants exhibited higher abundance of Ruminococcaceae associated with coconut-and rice-based dishes; and ethnic Chinese participants had greater levels of Bacteroides associated with seafood- and meat-rich diets. These ethnicity-diet-microbiome relationships were further corroborated by additional data from two independent Malaysian cohorts (n=544), a cohort from the United States (n=210), and an in vitro microbial culture model showing selective Bifidobacterium expansion by a fermented rice-based batter. Analysis of fecal microbiome-based risk scores revealed ethnic gradients in colorectal neoplasia scores that mirrored population-level cancer incidence patterns. Together, these findings characterize gut microbiome variations across major Asian ethnicities residing in a shared urban environment, providing a reference for precision health strategies relevant to over two billion ethnically relevant people in the Asia-Pacific region and beyond.

Streptococcus pneumoniae remains a leading cause of disease despite widespread vaccination, highlighting the need for serotype-independent strategies. We recently identified commensal streptococci that produce bacteriocins with anti-pneumococcal activity. Here, we evaluate these bacteriocins as candidates for pneumococcal control. Using cell-free protein synthesis, we screened 58 bacteriocins, the majority of which absent in available pneumococcal genomes, and identified SMiTE as the most potent. Purified SMiTE disrupted pneumococcal membrane integrity, as shown by confocal, transmission, and scanning electron microscopy, induced ATP leakage, and triggered transcriptional responses consistent with envelope stress and metabolic remodeling. In a mouse nasopharyngeal colonization model, intranasal SMiTE treatments reduced pneumococcal loads by 65-fold with no significant weight loss. SMiTE inhibited a broad range of serotypes, with strongest activity against serotype 3, which is poorly controlled by current vaccines, while sparing most oral and upper respiratory tract commensals. Repeated sub-inhibitory exposure did not select resistant mutants in line with the membrane-targeting mechanism. These findings establish SMiTE as a commensal-derived strategy for serotype-independent, microbiota-sparing pneumococcal decolonization.

Staphylococcus aureus is a leading cause of skin and soft tissue infections, yet colonization of healthy skin is limited by multiple defenses, including antimicrobial fatty acids (AFAs) produced by host cells and the resident microbiota. The mechanisms by which S. aureus overcomes these lipid-based defenses remain incompletely understood. Here, we show that mutations truncating the essential stringent response regulator Rel confer broad tolerance to both host-and microbially-derived AFAs in diverse S. aureus strains. Unlike classical stringent response activation, C-terminal Rel truncations do not induce typical (p)ppGpp-dependent transcriptional changes but instead enhance activity of the alternative sigma factor SigB and the staphylococcal accessory regulator SarA. This SigB-SarA regulatory cascade promotes transcriptional remodeling, including upregulation of pyrimidine biosynthesis genes, and coincides with alterations to cell envelope structure. Moreover, in the absence of SigB or SarA, tolerance can be restored through mutations in the serine/threonine phosphatase Stp1, highlighting additional pathways that modulate cell envelope-mediated resistance to AFAs. These findings identify a previously unrecognized consequence of Rel mutation in S. aureus; whereby small truncations may promote survival in AFA-rich host environments facilitating skin colonization and infection. ImportanceStaphylococcus aureus is a major cause of skin and soft tissue infections, and persistent skin colonization is a significant risk factor for infection. Antimicrobial fatty acids (AFAs), produced by microbes and host cells on human skin, normally limit S. aureus colonization. The mechanisms that allow this pathogen to overcome these lipid defenses are incompletely understood. We show that mutations truncating the essential stringent response regulator Rel enable S. aureus to tolerate both host-and microbially derived AFAs. These Rel variants, which have been identified in clinical isolates, alter cell envelope properties through the transcriptional regulator SarA rather than activating a classical stringent response. Our findings reveal a previously unrecognized adaptation that may facilitate S. aureus survival on the skin and promote infection.

Vitamin A is a central regulator of intestinal adaptive immunity, but its role in innate immunity is less defined. Antimicrobial proteins form a chemical barrier that protects the intestinal epithelium from microbial invasion. Among these, REG3 family lectins are induced by the microbiota, yet how nutritional cues intersect with microbial signals to control their expression remains unclear. Here, we show that dietary vitamin A promotes expression of REG3 antimicrobial lectins, including REG3G, in intestinal epithelial cells from both mice and humans. This induction is mediated by retinoic acid and requires retinoic acid receptor (RAR) signaling. Mechanistically, RARs bind directly to the Reg3g promoter adjacent to a STAT3 binding site. As STAT3 mediates microbiota-induced IL-22 signaling in epithelial cells, this arrangement provides a molecular framework for integrating nutritional and microbial inputs at the level of REG3G transcription. Extending these findings, we demonstrate that vitamin A-retinoic acid signaling similarly promotes expression of -defensin antimicrobial proteins. Together, these findings define a transcriptional mechanism by which vitamin A enhances epithelial antimicrobial defenses and strengthens mucosal innate immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/710399v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@18dd8aforg.highwire.dtl.DTLVardef@18cf2fdorg.highwire.dtl.DTLVardef@a4dc89org.highwire.dtl.DTLVardef@18aa9c7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIVitamin A promotes epithelial expression of REG3 antimicrobial proteins in the intestine C_LIO_LIRetinoic acid receptors (RARs) directly activate mouse Reg3g and human REG3G transcription C_LIO_LIRARs bind the Reg3g promoter adjacent to STAT3, integrating nutritional and microbial signals C_LIO_LIVitamin A-RAR signaling broadly regulates epithelial antimicrobial programs, including -defensins C_LI

Root-knot nematodes are obligate plant parasites that cause substantial agricultural losses worldwide. They induce highly specialized, metabolically hyperactive feeding sites within host roots, which serve as their sole source of nutrients throughout their life cycle. The formation and maintenance of these feeding sites depend on the manipulation of host developmental pathways by nematode-derived secretions. Phytosulfokines (PSKs) are small plant peptide hormones that regulate cell division, tissue expansion, and growth responses, processes essential for feeding site development. Here, we identify root-knot nematode genes predicted to encode peptides with a conserved PSK functional motif. These genes are predominantly expressed during the early stages of infection and localize to secretory glands, suggesting a role in early parasitism. Moreover, silencing PSK-like gene expression reduces root gall formation and nematode reproduction. Together, these findings reveal that root-knot nematodes deploy PSK-like peptides as virulence factors to promote successful parasitism, providing the first report of PSK peptide mimicry in any plant pathogen.

Aspartate represents an important proteogenic amino acid in all living organisms. Many microorganisms can produce aspartate through various biosynthetic processes, utilizing it for energy production and as a precursor for synthesizing other biomolecules, such as amino acids and nucleotides. The enteric pathogen Salmonella Typhimurium (S. Tm) has developed mechanisms to access aspartate as a nutrient source during expansion in the inflamed gut. However, how S. Tm deals with aspartate starvation during infection remains unknown. To address this knowledge gap, we interrogated Salmonellas reliance on the bi-directional aspartate aminotransferase encoded by aspC for growth in vitro and during host colonization using murine models of Salmonella infection. AspC can interconvert aspartate and the TCA intermediate oxaloacetate and is hypothesized to support S. Tm cellular demands for aspartate during starvation or support refueling of the TCA cycle via oxaloacetate synthesis. Herein, we find that loss of aspC results in a gut-specific S. Tm colonization defect that increases with the course of infection. Importantly, aspC is dispensable for S. Tm systemic colonization in CBA/J mice. Additionally, we report that loss of aspC results in a significant growth defect during respiration of inflammation-derived electron acceptors in vitro. Interruption of oxidative TCA cycle progression via TCA enzyme deletion or supplementation with TCA intermediates (e.g., oxaloacetate) abrogates the defect observed in {Delta}aspC S. Tm in vitro. Thus, suggesting the requirement for AspC to catabolize aspartate and support the TCA cycle during respiration. Altogether, we report that AspC plays a critical role in S. Tm pathogenesis in a gut-specific manner during inflammation through supporting energy generation.

De novo assembly of ancient and modern bacterial metagenomes can shed light on evolution and ecology of bacterial species that are challenging to culture. Tannerella and Porphyromonas are bacterial genera linked to periodontal disease, and understanding their evolution may reveal insights into their role in oral disease development. We performed pangenomic and phylogenetic analyses on a global set of isolates and metagenome-assembled genomes of the genera Tannerella (n=238) and Porphyromonas (n=976), including 66 genomes from ancient dental calculus samples (up to 14,800 years old), and modern oral samples from present-day living populations. We identify a novel species of oral Tannerella in modern and ancient humans, which we call Ca. Tannerella abscondita, that is related to and often mistaken for Tannerella forsythia but differs in its virulence repertoire. We reveal distinct niche tropism in Tannerella species and Porphyromonas pasteri, but not Porphyromonas gingivalis. There is limited phylogeographic structuring, and virulence genes are homogeneously distributed across continents and oral niches. Saliva-derived strains of T. forsythia and P. gingivalis from Oceania and T. serpentiformis and P. pasteri from Asia show enrichment of pseudogenes related to ecological niche transitions. A phylogenetic analysis of the P. gingivalis major fimbrial protein gene fimA reveals the genes cluster by genotypes, and that no ancient genes are found in genotypes I and Ib. Using de novo assembly for bacterial pangenomics improves the representation of oral genera found in reference databases and enhances our ability to study the evolutionary history of these taxa. Significance statementPangenome analyses have been primarily applied to clinically significant species that are abundant and easy to culture, leaving a substantial number of human commensal microbes uninvestigated. De novo assembly of commensal microbial metagenomes is a significant resource for generating metagenome assembled genomes (MAGs) of difficult-to-culture species, which can be used for pangenome analyses. We investigated the ecological niches and geographic localization of multiple species of the human oral commensal genera Tannerella and Porphyromonas using MAGs assembled from globally diverse modern and ancient populations. While much work on these genera comes from Europe and North America, we note distinctions in ecological niches and species prevalence globally, highlighting how MAGs overcome limitations set by culture-based approaches that dominate currently available data. Additionally we demonstrate how improved taxonomic representation of diverse oral species through MAGs can clarify the evolutionary history of commensals and their virulence factors associated with oral disease.

During evolution, bacteria have developed the ability to interact intimately with eukaryotic hosts. These interactions span a dynamic continuum ranging from pathogenicity to mutualism, along which bacteria can rapidly evolve and shift their lifestyles. However, the molecular mechanisms that enable bacteria to adapt to new hosts and to transition between distinct interaction modes remain poorly understood. Here, using a unique combination of two independent evolution experiments, we identified and characterized parallel adaptive mutations in spoT, which encodes the bifunctional (p)ppGpp synthetase-hydrolase. These mutations promote the adaptation of the plant pathogen Ralstonia pseudosolanacearum to two distinct plant-associated environments and two distinct lifestyles, the xylem of both susceptible and tolerant host plants as a pathogen and the root nodules of a legume as a symbiont, without compromising virulence on susceptible hosts. These mutations enhance the utilization of multiple carbon and nitrogen sources, including substrates known to be abundant in xylem sap, and increase bacterial exponential growth rate in minimal medium, suggesting reduced basal (p)ppGpp levels. Assessment of a strain deficient in SpoT synthetase activity confirmed that lowering basal (p)ppGpp levels is adaptive in both plant environments. Together, our findings reveal that fine-tuning intracellular (p)ppGpp concentrations represents an efficient strategy for optimizing bacterial adaptation to complex host-associated environments.

Monosaccharides support Salmonella enterica serovar Typhimurium colonization of the gut, yet the role of their oxidized derivatives remains understudied. Sugar acids are largely diet-independent carbon sources generated by host-driven oxidative processes, but their contribution during infection - particularly that of less oxidized aldonic and uronic acids - has not been defined. Here, we systematically assess the role of sugar acids derived from D-glucose and D-galactose in S. Typhimurium SL1344 colonization. Among D-glucose-derived acids, D-gluconate accumulated to the highest levels and was the dominant substrate supporting luminal expansion in streptomycin-pretreated mice, exceeding the more oxidized acids D-glucuronate and D-glucarate. During chronic infection, D-glucose-derived sugar acids became increasingly important for pathogen persistence. Ecological niche invasion assays identified these compounds as a principal metabolic niche, whereas D-galactose-derived acids contributed minimally. Consistent with a transient, inflammation-linked nutrient niche, sugar acid utilization pathways were similarly prevalent in Escherichia coli from individuals with and without inflammatory bowel disease. Together, these findings identify D-gluconate as a key inflammation-dependent nutrient source that fuels Enterobacteriaceae expansion in the inflamed gut.