Gene Cluster Research Articles (Page 1)

In this study, we present a comprehensive characterization of a highly efficient desulfurizing bacterial isolate, SB1D. The isolate exhibited remarkable desulfurization of dibenzothiophene (DBT) and demonstrated the ability to metabolize benzothiophene (BT) and several of their alkylated derivatives. Genome-related index analyses, including 16S rRNA gene similarity (100%), Average Nucleotide Identity (ANI; 98.7%), digital DNA-DNA hybridization (dDDH; 88.7%), and phylogenomics, identified the strain as Rhodococcus qingshengii. Additionally, orthologous gene cluster analysis showed that SB1D shared the highest number of ortholog clusters (60) with R. qingshengii. The GC-MS analysis of the extracted metabolites identified 2-hydroxybiphenyl (2-HBP) and 4-methylhydroxybiphenyl (4-MHBP) as the major end-products of DBT and 4-methyldibenzothiophene (4-MDBT) desulfurization, respectively. The RAST genomic analysis revealed the presence of several organic-sulfur metabolism-related genes in the genome of SB1D. Together, these findings confirm that the isolate employs the sulfur-specific 4S pathway for the desulfurization of DBT and 4-MDBT. To our knowledge, this is the first report providing genome-based characterization and desulfurization pathway analysis of R. qingshengii SB1D, with the proven ability to desulfurize multiple thiophenic compounds found in diesel, and holds promise as a valuable biocatalyst for applications in biodesulfurization.

Rice produces a diverse array of phytoalexins, including diterpenoid compounds (momilactones and phytocassanes) and the flavonoid sakuranetin, which serve as crucial defense metabolites against environmental stresses such as pathogen attack. This review summarizes the regulatory mechanisms and evolutionary insights of rice phytoalexin biosynthesis. Jasmonoyl-l-isoleucine (JA-Ile) is one of the signal molecules inducing phytoalexin production. OsCOI2 functions as the primary JA-Ile receptor for phytoalexin production. Multiple transcription factors, including DPF/bHLH25, OsTGAP1, and various WRKY proteins, coordinately regulate the expression of biosynthetic genes. Remarkably, genes encoding diterpenoid phytoalexin biosynthetic enzymes are organized into biosynthetic gene clusters in the rice genome. Comparative genomic analyses reveal dynamic evolutionary processes involving gene duplications, cluster rearrangements, and occasional losses across Oryza species. These findings provide fundamental insights into the evolution of plant chemical defense and offer potential strategies for developing stress-tolerant crops by targeting the manipulation of phytoalexin biosynthetic pathways and their regulatory networks.

The marine pathogen Yersinia ruckeri synthesizes the tri-catecholate siderophore ruckerbactin, Rb, (DHB-LArg-LSer)3, to acquire iron during infection. Its biosynthetic gene cluster encodes a single periplasmic binding protein, RupB, which surprisingly does not bind Fe(III)-Rb nor the Fe(III) complexes of its hydrolysis products, the di- and mono-catecholate siderophores RbDC and RbMC, with biologically relevant affinities. Instead, the periplasmic binding protein YiuA, encoded in a different region of the chromosome, binds the 1 : 2 Fe(III) complex of the mono-catecholate RbMC, Fe(III)-(RbMC)2. YiuA is the first periplasmic binding protein (PBP) to selectively recognize a mono-catecholate siderophore, the structural basis of which was illuminated through X-ray crystallography of YiuA bound to Fe(III)-(RbMC)2.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are remarkable natural products with interesting chemical structures and potent bioactivities. RiPP pathways are abundant in all domains of life and harbor a large biosynthetic potential in the form of post-translationally acting enzymes. A relatively small number of RiPP biosynthetic gene clusters encode peptide arginases, a recently discovered maturase family capable of hydrolyzing arginine residues of RiPP core peptides to ornithines. In this study, members of the peptide arginase family (FlmR and OhkR), which are associated with uncharacterized precursors from orphan RiPP families, are identified. In vivo and in vitro activity of FlmR and OhkR with the five associated precursor peptides (FlmA1-3 and OhkA1-2) is demonstrated and kinetic studies to biochemically characterize the enzymes are performed. Furthermore, in silico structural analysis with AlphaFold 3 is used to predict precursor-arginase complexes, providing insights into how peptide arginases could bind their precursor substrates. In the case of OhkA-OhkR complexes, this analysis also allows a hypothesis as to which of the arginine residues of the core peptide is modified first, which is confirmed experimentally. This detailed biochemical and structural enzyme characterization is a prerequisite for the application of peptide arginases in peptide-based drug discovery platforms.

Pepper southern blight, caused by Agroathelia rolfsii (Sacc.) Redhead & Mullineux (syn. Sclerotium rolfsii Sacc.), is a serious soil-borne fungal disease. The overuse of chemical pesticides to control this disease has led to pathogen resistance and environmental pollution, making biological control methods a more sustainable alternative. In this study, a strain SEC-482 with significant antagonistic effect against A. rolfsii was isolated from the rhizosphere soil of peppers. It was identified as Bacillus velezensis through morphological, physiological, biochemical and molecular analyses. The strain showed a high inhibition rate of 76.44% ± 0.37% against A. rolfsii in vitro and a control effect of 72.73% on pepper southern blight in pot experiments. Furthermore, it was observed to have a favourable impact on the growth of pepper plants. The genome sequencing and analysis revealed many genes related to antibiosis and growth promotion, as well as 14 secondary metabolite synthesis gene clusters. The strain's volatile organic compounds (VOCs), such as 2,6-diethylpyrazine, isobutyric acid and 3,4-diaminophenol, 3,5-dimethyl-1-prop-2-ynyl-pyrazole, were identified as the main antimicrobial substances. This study demonstrates that B. velezensis SEC-482 has potential for the biological control of pepper southern blight, laying a foundation for subsequent field trials to confirm its practical application value. The identification of VOCs and the exploration of the strain's fermentation conditions provide valuable insights into its potential applications in sustainable agricultural practices.

Exposure to maternal type 1 diabetes (T1D) during pregnancy provides relative protection against T1D in the offspring. This protective effect may be driven by epigenetic mechanisms. Here we conducted an epigenome-wide blood analysis on 790 young children with and 962 children without a T1D-affected mother, and identified differential DNA methylation (q < 0.05) at multiple loci and regions. These included the Homeobox A gene cluster and 15 T1D susceptibility genes. The differential methylation was found in transcriptionally relevant regions associated with immune function, including sites previously linked to T1D-related methylation loci and protein biomarkers. Propensity scores for methylation at T1D susceptibility loci could predict the development of islet autoimmunity in offspring born to mothers without T1D. Together, these findings highlight pathways through which maternal T1D may confer protection against islet autoimmunity in offspring and suggest that environmental factors can influence T1D risk through epigenetic modifications of T1D susceptibility loci.

Endophytic bacteria are endosymbiotic bacteria that colonize the inter- or intracellular regions of plants, providing them with protection through cross-communication and the release of metabolites via their biosynthetic gene clusters. The emergence of new diseases due to rapid multidrug resistance against existing drugs has created an indisputable need for biologically active secondary metabolites. This study aimed to assess the antibacterial activity of secondary metabolites from endophytic bacteria isolated from Senna occidentalis against multidrug-resistant clinical bacterial isolates. Endophytic bacteria were isolated from the leaves, stems, and roots of S. occidentalis (coffee senna) and identified based on their cultural, microscopic, and biochemical characteristics. The Agar well diffusion method was employed to evaluate the endophytes for antibacterial activity against clinical isolates of Staphylococcus aureus, Salmonella Typhi, and Pseudomonas aeruginosa. A total of twenty-three endophytes were isolated from Senna occidentalis, and molecular identification using the 16S rRNA gene sequencing method was carried out for the three most active endophytic bacteria, each from the leaf, stem, and root of the plant, respectively. Screening using agar well diffusion method yielded mean inhibitory zones ranging from 30.00±0.00 (mm) to 12.00±0.00 (mm) for the secondary metabolites. Molecular identification revealed 99–100% sequence identity to related type strains in two major classes: Gammaproteobacteria (Escherichia sp.) and Bacilli (Bacillus sp.). The findings of this study suggest that endophytic bacteria from S. occidentalis can serve as a promising source of novel antimicrobial compounds with broad therapeutic significance.

The Bacillusgenus comprises spore-forming, Gram-positive bacteria widely recognized for their capacity to produce bioactive compounds with antimicrobial properties. This activity is primarily attributed to the synthesis of diverse molecules, including peptides, non-ribosomal peptides, and polyketides, which exhibit inhibitory effects against various pathogens. Bacillus species are ubiquitous and highly diverse, encompassing strains with significant biotechnological potential. Bac-9 is a bacterial strain belonging to Bacillus genus isolated from kefir in Escárcega, Campeche, México. This strain exhibits strong antifungal activity against Fusarium equiseti, F. solani, Curvularia sp. and the oomycete, Phytophthora capsici. The genome comprises 4,059,427bp, with a total of 4,195 coding sequences and a GC content of 46.3%. A total of 21 biosynthetic gene clusters were identified: 55% were non-ribosomal peptides (NRPs), 36% were polyketides (PKs) and 9% were NRP + PK hybrids. This bacterium produces compounds with insecticidal properties, such as thietane and acenaphthylene, which were detected by GC-MS. In tomato plants, different Bac-9 extracts activate genes involved in defense against insects and pathogens, including genes associated with the biosynthesis of jasmonic acid and salicylic acid. In addition, the extracts reduce oviposition by the whitefly Bemisia tabaci in Lycopersicon esculentum. This study offers valuable insights into bioactive compounds with potential for the biological control of phytopathogens and whiteflies. Overall, the bacterium emerges as a promising candidate for use in biological control strategies.

Bisphenol F (BPF) is an emerging environmental pollutant widely present in surface water and wastewater systems. Microbial activity is crucial in driving its degradation, offering a potential avenue for mitigating its environmental impact. Although the degradation pathway for BPF has been identified in various bacteria, the biodegradation mechanisms remain unclear. In this study, we isolated a highly efficient BPF-degrading strain of Sphingobium yanoikuyae DN12, which could utilize BPF as the sole carbon and energy source for growth, from a river sediment in Anhui Province, China. Through ultra-performance liquid chromatography high-resolution mass spectrometry (UPLC-HRMS) analysis, we found that oxidation and hydrolysis are key steps for BPF biodegradation. Utilizing whole-genome sequencing, comparative transcriptomics analysis, and biochemical identification, a gene cluster bpf was identified to be involved in BPF degradation. BpfAB is a two-component oxidoreductase responsible for converting BPF to 4,4'-dihydroxybenzophenone (DHBP). BpfC is a Baeyer-Villiger monooxygenase responsible for converting DHBP to 4-hydroxyphenyl-4-hydroxybenzoate (HPHB). Isotope tracing demonstrated that the oxygen atom incorporated by BpfAB originates from water, whereas that incorporated by BpfC derives from molecular oxygen (O2). BpfD is an α/β hydrolase responsible for converting HPHB to 4-hydroxybenzoate and 1,4-hydroquinone. Analysis of the taxonomic and habitat of 325 prokaryotic genomes revealed that BpfA-like homologs are predominantly found in the phylum Pseudomonadota, primarily inhabiting soil and aquatic environments. This study enhances our understanding of the biodegradation mechanism of BPF and provides guidance for the effective remediation of BPF-contaminated environments.IMPORTANCEBisphenol F (BPF) is a widely used alternative to bisphenol A and poses a growing threat to ecosystems and human health due to its environmental persistence and endocrine-disrupting effects. Although microbial degradation pathways for BPF have been reported, the key enzymes involved and their catalytic mechanisms remain unclear. This work reports the isolation of a Sphingobium strain capable of mineralizing BPF and the genetic basis for the catabolic pathway. Three enzymes-a two-component oxidoreductase, a Baeyer-Villiger monooxygenase, and an α/β hydrolase-were biochemically characterized and shown to catalyze the three critical steps in BPF degradation. These findings provide insights into the biochemical processes involved in the microbial degradation of BPF.

The Serrated Hinge-back Tortoise Kinixys erosa inhabits moist forests across Central and West Africa and is known to show phylogeographic structure. Based on extended geographic sampling, we re-examined its phylogeography using three mitochondrial genes and up to 17 nuclear loci. The observed mtDNA variation was considerable and corresponds to two major and well-supported clades from the western and the eastern part of the distribution range. Within the western clade, samples from Ghana represent a well-supported subclade. Nuclear loci support the genetic distinctness of these groups showing the Ghanian population as the most divergent. This suggests that K. erosa comprises hitherto unrecognized distinct taxa. Since no sufficient morphological data are available, and it is unclear to which clade the name K. erosa (Schweigger) refers, we abstain from taxonomic conclusions, but identify the genetic clusters as distinct Management Units for conservation.

Background: Porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) remains a significant threat to swine production globally, including Japan. While the genetic diversity of PRRSV-2 has been reported previously, the potential association with modified live vaccines (MLVs) is not well understood. This study aimed to characterize PRRSV-2 strains currently circulating in Japan and assess possible links with MLVs. Methods: A total of 1190-nucleotide open reading frame 5 sequences of PRRSV-2 were collected across Japan between 2020 and 2023, and phylogenetic analyses were performed to classify genetic clusters. Additionally, correlations between cluster distribution and MLV usage were examined, using sequences detected in the Kanto region. Results: Phylogenetic analysis revealed that 48.5% of the sequences belonged to Cluster III, with a median nucleotide identity of 88.2% to the Japanese reference strain EDRD-1. Notably, the sequence identity between the strains detected in this study and EDRD-1 was significantly lower than that of strains identified in 1992–1993 (p &lt; 0.05). In the Kanto region, Cluster I and II variants, which exhibited high sequence homology to MLV strains, were exclusively detected on farms with a history of MLV usage. Furthermore, Cluster IV displayed substantial genetic divergence, suggesting it comprises a heterogeneous group of distinct lineages. Conclusions: These findings demonstrated the temporal changes in the genetic diversity of Cluster III and provided suggestions of a possible influence that MLV usage influences PRRSV-2 cluster distribution, with Clusters I and II likely representing vaccine-origin viruses. The marked heterogeneity of Cluster IV also highlights the limitations of the current cluster-based classification.

Fusarium wilt, caused by Fusarium oxysporum f. sp. vasinfectum 7 (FOV7), poses a major threat to the production of elite Sea Island cotton ( Gossypium barbadense ). To uncover the molecular basis of defense FOV7 in cotton, we employed RNA sequencing to identify numerous differentially expressed genes across various stages of infection. Subsequent K-means clustering and weighted gene co-expression network analysis revealed a core module significantly enriched in defense response and abscisic acid-activated signaling pathways. A detailed examination of the gene distribution within these pathways identified 10 out of 50 genes as members of the Pathogenesis-Related 10 ( PR10 ) gene family. Evolutionary analysis of these PR10 genes uncovered a tandemly-expanded gene cluster located on chromosome 10 of the D sub-genome. In addition, root cell type maps constructed via single-nucleus RNA sequencing (snRNA-seq) enabled pinpointing FOV7 response in the root epidermis, where GbD_PR10.11 was identified as a specifically activated sentinel. Our work, by logically progressing from genome-wide patterns to a single gene in a single cell type, not only deciphers a key component of the cotton-pathogen arms race but also delivers a high-confidence target for engineering frontline resistance.

More than 80% of clinical antibiotics are compounds produced by soil bacteria, and there are many more unknown pathogen-inhibiting compounds encoded by clusters of co-localized genes (i.e., biosynthetic gene clusters) in soil bacterial genomes. To find novel antimicrobial drug candidates using a genome-level approach, biosynthetic gene clusters (BGCs) can be identified and compared against a database of known BGCs to infer what secondary metabolite(s) may be encoded. This study aims to uncover the diversity of these BGCs and characterize evolutionary patterns of BGC distribution across a phylogeny of 305 soil-derived bacterial isolates. BGCs were predicted through antiSMASH and taxonomic classification was conducted via GTDB-tk. OrthoFinder was used to perform a multi-locus sequence analysis and phylogenetic tree construction. The results of this investigation, an annotated phylogeny with mapped BGC data, will provide future antibiotic discovery researchers with deeper genetic insight into the biosynthetic potential and evolutionary patterns of BGCs for the investigated bacterial strains.

Background Raoultella ornithinolytica is an infrequent opportunistic pathogen capable of causing multi-site infections and frequently harboring a broad array of resistance determinants, thereby complicating antimicrobial therapy. Here we report the genomic characterization of the extensively drug-resistant strain FAHZZU6693, which concurrently harbors bla NDM-1 , bla KPC-2 , mcr-10 genes and tmexCD2-toprJ2 resistance cluster. Methods Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and average nucleotide identity (ANI) were employed to confirm the species identity as R. ornithinolytica . Antimicrobial susceptibility testing (AST) delineated the corresponding antimicrobial phenotypes. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting and whole-genome sequencing (WGS) elucidated the isolate’s complete molecular architecture. Results Globally, R. ornithinolytica strains harboring related resistance genes exhibit diverse geographical distribution. Strain FAHZZU6693 is resistant to most antibiotics, except amikacin and chloramphenicol. The bla NDM-1 , bla KPC-2 , mcr-10 genes and the tmexCD2-toprJ2 cluster in this strain are plasmid-borne. These occur in conserved genetic contexts: xerC-mcr-10- IS Ec36- IS Ec27- IS Ecl1 and umuC- IS 881-tmexC2-tmexD2-toprJ2-umuC . Further analysis indicates that the insertion sequence IS Ec27 and the gene element umuC play a crucial role in the dissemination of the mcr-10 gene and the tmexCD2-toprJ2 gene cluster. Conclusions This study combines database analysis to comprehensively describe the distribution of R. ornithinolytica strains carrying the target genes and characterizes the genomic features of a clinically Multi-drug resistant strain, providing a theoretical foundation for preventing the spread of such bacteria.

Tetracladium spp. represent a group of fungi that inhabit various ecological niches, including soil and aquatic environments, where they are considered to have a saprotrophic lifestyle and within plant roots as endophytes. To date, a lack of sequenced Tetracladium spp. genomes has inhibited our understanding of their metabolic potential and ecological interactions. In this study, we aimed to elucidate the genetic differences between aquatic saprotrophic and endophytic strains of Tetracladium spp. by sequencing and analysing the genomes of T. maxilliforme (isolated from Brassica napus roots) and T. marchalianum (isolated from freshwater), alongside 41 publicly available saprotrophic and endophytic Ascomycetes. Genomic sequencing revealed that T. maxilliforme possesses a genome size of 35.5 Mbp with 9657 predicted genes, while T. marchalianum has a genome size of 33.2 Mbp with 15,230 predicted genes. Our analyses primarily focused on carbohydrate-active enzymes (CAZymes). Both genomes possessed the full range of enzymatic machinery for cellulose degradation, as well as the complete repertoire of genes necessary to degrade plant cell walls. Notably, the genomes lacked essential enzymes for lignin degradation or modification. Furthermore, we observed a complete repertoire of known fungal chitin-degrading enzymes in both genomes, which might be related to potential interactions with other fungi. Enzyme composition profiles revealed distinct groupings, with T. maxilliforme primarily clustering with endophytic or ecologically versatile species, while T. marchalianum was predominantly associated with saprotrophic species. We also identified secondary metabolite biosynthetic gene clusters in both genomes, including several that showed high homology to those of known bioactive compounds. In summary, our findings offer valuable insights into the genomic adaptations of Tetracladium spp. to various ecological niches, highlighting their enzymatic capabilities for carbohydrate degradation and potential interactions within fungal communities.

Aspergillic acid, a hydroxamate-containing pyrazinone derived from leucine and isoleucine, is a hallmark metabolite of Aspergillus flavus, which also produces the potent carcinogen aflatoxin. Its biosynthesis is governed by the recently characterized asa gene cluster. While A. flavus consistently produces aspergillic acid, its domesticated relative Aspergillus oryzae has long been considered a non-producer. Here, we compared the asa cluster activity and metabolite output in A. flavus NRRL 3357 and two A. oryzae strains (RIB40 and NRRL 3483) under amino acid-rich conditions. In A. flavus, asa genes were strongly induced in casein peptone medium, resulting in robust production of aspergillic acid, deoxyaspergillic acid, and iron-chelating ferriaspergillin. A. oryzae RIB40 showed no transcriptional activation or metabolite production. By contrast, A. oryzae NRRL 3483 exhibited clear asa cluster induction and detectable accumulation of deoxyaspergillic acid and trace aspergillic acid sufficient to form ferriaspergillin. Notably, the disproportionate buildup of deoxyaspergillic acid revealed a bottleneck in downstream tailoring steps. These results demonstrate that A. oryzae retains strain-specific, conditionally inducible aspergillic acid biosynthesis, highlighting evolutionary attenuation of secondary metabolism in domesticated fungi. This work establishes a framework for dissecting regulatory and enzymatic constraints in the asa pathway, highlighting the potential of hidden biosynthetic clusters for natural product discovery and biotechnological applications.

Abstract Habitat loss due to deforestation and overexploitation are major causes threatening the long-term survival of Amentotaxus yunnanensis, a vulnerable conifer native to Vietnam. To establish a genetic baseline for conservation, we assessed 222 individuals from eight populations using nine polymorphic microsatellite (SSR) markers. The analysis revealed moderate species-level genetic diversity (mean expected heterozygosity HE = 0.353) but strong genetic differentiation among populations (FST = 0.122). This structure is driven by a historical pattern of isolation by distance, which is now severely compounded by recent habitat fragmentation that restricts gene flow. Population structure analyses consistently identified two distinct genetic clusters corresponding to Northern and Central Vietnam. Furthermore, bottleneck analysis revealed recent demographic declines in the four northernmost populations, three of which simultaneously retain the high levels of genetic diversity. These findings define two distinct management units (MUs) with disparate conservation needs. The declining northern populations are critical reservoirs of genetic diversity requiring urgent in situ protection, while the genetically depleted central populations necessitate habitat connectivity strategies. Our results provide an essential framework for developing targeted conservation actions to ensure the persistence of this threatened species.

Abstract Walnut biodiversity represents a critical genetic resource for developing climate-resilient cultivars capable of withstanding increasing drought stress under changing environmental conditions. Understanding the genetic basis of drought tolerance mechanisms is essential for sustainable nut production and conservation of valuable genetic resources. This study investigated the genetic and physiological basis of drought tolerance in walnut ( Juglans regia L.) saplings, providing crucial insights for breeding programs aimed at developing climate-resilient cultivars. A comprehensive analysis was conducted using 65 walnut samples collected from diverse geographic locations, employing SSR (simple sequence repeat) markers for genetic characterization combined with detailed morphological and physiological assessments under controlled drought stress conditions. Our findings revealed significant variability in drought stress responses among walnut saplings. Morphological and physiological traits exhibited substantial variation under drought stress, reflecting the genetic diversity present in natural populations. Population structure analysis performed using STRUCTURE software identified three distinct genetic clusters within the studied population, with varying degrees of admixture indicating complex evolutionary relationships and gene flow patterns across different populations. Some individuals showed strong affiliation to a single cluster, while others exhibited significant admixture patterns. Mixed Linear Model analysis identified significant marker-trait associations between SSR markers and drought tolerance traits. Multiple markers showed strong correlations with physiological parameters including chlorophyll content, leaf characteristics, and stress response indicators. Hierarchical clustering and phylogenetic analyses confirmed the genetic relationships among populations and revealed patterns consistent with geographic distribution and environmental adaptation. This research elucidates the genetic architecture underlying drought tolerance in walnuts and demonstrates the effectiveness of SSR markers for identifying drought-responsive genes. The identified marker-trait associations provide valuable tools for marker-assisted selection in breeding programs. Furthermore, the observed genetic diversity and its correlation with drought-related traits offer a foundation for developing more resilient walnut cultivars. These findings contribute to the conservation and utilization of walnut genetic resources while supporting the development of climate-adaptive breeding strategies for sustainable nut production under water-limited conditions.

Introduction: Psychosocial stressors in early pregnancy are associated with a higher risk of adverse pregnancy outcomes (APOs), including hypertensive disorders of pregnancy (HDP), gestational diabetes, and preterm birth. However, the biological pathways underlying these associations are not well delineated. This study aims to investigate proteomic markers that may underlie the association between early pregnancy psychosocial stress and APOs. Methods: Data were from the Nulliparous Pregnancy Outcomes Study: Monitoring Mothers-to-Be, a prospective study conducted from 2010-13. Participants were selected using a case-control design (508 HDP cases and 1081 controls). An aptamer-based assay was used to quantify 6,894 proteins in blood serum collected at the first-trimester study visit. Psychosocial stress during the month preceding the same visit was defined as a score greater than 13 on the 10-item Perceived Stress Scale. We used linear regression, adjusted for age and gestational age, to estimate the associations between stress and proteomic analytes. We then used logistic regression models to estimate the associations of these analytes with APOs. To identify potential biological pathways, we constructed a knowledge graph integrating Human Phenotype Ontology terms and STRING protein-protein interactions. Results: Among 1,589 pregnant participants, the mean (SD) age was 27 (6) years and 42% reported psychosocial stress. Heparan sulfate 6-O-sulfotransferase 3 (HS6ST3) was significantly associated with both stress and APOs after FDR correction. Higher psychosocial stress was associated with lower expression of HS6ST3 (-0.27 [95% CI -0.32, -0.22]). Also, a lower expression in HS6ST3 was associated with higher risk of HDP (aOR 0.72 [0.61, 0.85]), gestational diabetes (aOR 0.59 [0.48, 0.71]), and preterm birth (aOR 0.75 [0.61, 0.92]). Proteomic values were expressed in SD units of log2-transformed SomaScan measurements. A knowledge graph was created, which identified close connections between anxiety (phenotype term closest to stress), APOs, and HS6ST3 with shared biological pathways, including GPC* gene clusters, SHH, LYN, and PTCH1 ( Figure ). Conclusions: Early pregnancy psychosocial stress was significantly associated with lower HS6ST3 expression and increased risk of APOs. Genes known to interact with HS6ST3, which are involved in neuroimmune signaling, placental development, and vascular function, may represent plausible pathways linking psychosocial stress to APOs.

The anaerobic bacterium Clostridium perfringens is commonly found in the intestinal tract of humans and animals. However, there are marked differences in virulence between isolates and toxinotypes, which largely depend on various virulence factors produced by these strains. Studying C. perfringens genomes has been limited by fragmented assemblies from short-read sequencing and incomplete clinical metadata. Here, we present a high-quality collection of 236 isolates from animal hosts that underwent detailed pathomorphological examination. From 220 of them, genomes were generated by PacBio long-read sequencing, enabling comprehensive, structural level analysis of their virulome, plasmids, conjugative elements and biosynthetic gene clusters (BGCs). One hundred forty isolates were collected from animals with signs of C. perfringens-associated enteric disease, 32 from animals with no signs of C. perfringens-associated disease and 64 from healthy animals. An additional 383 publicly available C. perfringens complete or draft genomes were included for comparative analyses. We discovered 2 previously undescribed pore-forming toxin (PFT) homologues and 11 novel haemolysin and aerolysin-type PFT variants. Both findings expand the known spectrum of the C. perfringens virulome. Moreover, we defined two novel putative plasmid conjugative loci in a collection of 888 here assembled and circularized plasmids. They may facilitate HGT, supporting the dissemination of virulence and metabolic traits. We predicted 414 BGCs that were frequently toxinotype specific and encoded centrally for a bacteriocin that could help their carriers to outcompete other bacteria in shared environments. Furthermore, comparative analysis of C. perfringens plasmids revealed three distinct clusters based on their conjugation system. Altogether, these findings significantly expand the landscape of animal-associated C. perfringens through high-quality genome data and highlight novel virulence-associated features that provide a foundation for future studies of this important pathogen.