Genomics

Hurthle cell carcinoma (HCC) is an aggressive form of thyroid cancer. While mitochondrial DNA mutations and chromosomal losses have been identified in HCC, isoform switching, and its functional consequences remain uncharacterized. This study reanalyzed NCBI GEO dataset GSE228870 (n = 32), using Salmon and IsoformSwitchAnalyzeR() to identify isoform switching. The analysis resulted in 371 switches across 335 genes showing functional consequences including loss of protein domains, shorter open reading frames (ORFs), loss of signal peptides and novel sub-cellular localizations. Most significant isoform switches (q-value < 0.05, |dIF| > 0.1) were observed in LAMA2, LSP1, MAD2L2, FBLN2 and CXCL12, implicating extracellular matrix dysregulation, DNA damage response, immune signaling and cytoskeleton regulation. These genes are expressed in normal thyroid (median TPM 20.69, 11.66, 14.79, 134.1 & 80.76). However, specific isoforms of LAMA2 and MAD2L2 are not expressed in normal thyroid, explaining tumor-specific expression in HCC. Alternative transcription termination site (ATTS) gain was significant, suggesting altered 3 end in HCC transcripts. TCGA SpliceSeq showed LSP1, FBLN2 and CXCL12 undergo alternative promoter (LSP1 exon1 PSI=94.5%, FBLN2 exon2 PSI=99.0%) and alternative termination (CXCL12 exon3.3 PSI=53.9%) in thyroid cancer, suggesting ATTS and alternative transcription start site (ATSS) as shared splicing dysregulation mechanisms. This is the first systematic characterization of isoform-level dysregulation in HCC.

We applied the latest RIC-seq technology to study the RNA-RNA interactions in human liver HepG2 cells. Integrating ChIP-seq data with the RIC-seq data, we clas-sified vari ous transcription factors (TFs) into two distinct clusters based on the in-teraction enrichment scores of the uaRNA (RNA transcribed in promoter regions) and eRNA (RNA tran scribed in enhancer regions) potentially transcribed by the TF binding sites. We went on to compare the genomic features and clinical significance across different clusters, by further integrating Hi-C, ATAC-seq, and clinical data from liver cancer patients. Our results showed that Cluster 1 exhibited low spatial interaction (RIC-seq derived RNA-RNA interactions between regulatory RNA ele-ments) and high tissue specificity (high proportion of binding sites covered by shared peaks between two different cell lines for each histone modification marker), showed higher associations with the B compartments and nucleoli, contained less densely clustered TF binding sites, and is more correlated with clinical outcomes. In contrast, Cluster 2 has higher spatial interaction, is more likely to be found in the A compartments and nuclear speckles, contained more densely clustered TF binding sites, and is more prone to form loops. These findings suggested that these transcrip-tion factors might play critical roles in regulating gene expression and serve as po-tential biomarkers and therapeutic targets with significant implications for cancer prognosis and treatment. In summary, groups of TFs could occur as distinct sub-networks based on spatial proximity of non-coding RNAs transcribed by the TF binding sites. These sub-networks appear to be related to regulatory differences for the TF groups and may partially explain the chromosome compartments/domains.

The liver of laying hens plays a central role in metabolism and reproduction, supporting the synthesis of egg yolk precursors under strong hormonal regulation. Despite its physiological importance, a high-resolution cellular reference of the adult chicken liver is still lacking. Here, we generated a single-nucleus RNA sequencing atlas of the adult laying hen liver from eight individuals, providing a comprehensive view of its cellular composition and transcriptional landscape. Using this framework, we identified major hepatic cell populations, including hepatocytes, endothelial cells, cholangiocytes, hepatic stellate cells, and diverse immune cell types, revealing a broadly conserved vertebrate liver architecture. However, hepatocyte zonation, a key feature of mammalian liver organization, was not observed, consistent with the absence of hepatocyte zonation reported in birds. Importantly, we demonstrate that the use of an enriched genome annotation, incorporating additional protein-coding and long non-coding RNA models, substantially improves transcript detection and enhances cell-type resolution in single-nucleus datasets. This improved resolution allows more accurate marker-based assignment of hepatocyte subpopulations and refines the interpretation of hepatic cellular heterogeneity. Within hepatocytes, we uncovered transcriptionally distinct subpopulations associated with lipid metabolism and reproductive function, including estrogen-responsive programs involving cytochrome P450 genes such as CYP2C23A and CYP2C23B. In parallel, we characterized a complex immune compartment composed of resident macrophages and adaptive immune cells, highlighting the dual metabolic and immunological roles of the avian liver. Overall, this atlas provides a high-resolution reference for avian liver biology and demonstrates that improved genome annotation enhances the resolution and interpretation of cellular heterogeneity in single-cell transcriptomic studies.

Environmental and dietary factors can exert multigenerational effects on health and development. In this study, we investigated whether early-life metabolic challenge affects the germline genome and epigenome across three generations. Using a murine model of early life obesity via litter size reduction (overnutrition group, ON) and a control group (CT), we followed the paternal lineage focusing on germline genomic and methylation changes employing Genotyping-by-Sequencing (GBS) coupled with methyl-immunoprecipitation (GBS-MeDIP). We found that unrelated ON families clustered together based on identified Single-Nucleotide Polymorphism (SNP), suggesting that the treatment may have genomic impact. Copy number variations (CNVs) events were identified in ON individuals, being enriched in Long Interspersed Nuclear Elements (LINEs) and Long Terminal Repeats (LTRs). While Principal Component Analysis (PCA) of the methylome showed no clear treatment effect, pathway enrichment and regional analyses revealed methylation changes associated with transposable elements and developmental genes. Notably, the ON group exhibited a disruption in the methylation of Repetitive Elements (RE), which was significant in the same type of RE that were also enriched in the observed CNVs. The ON also showed reduced emergence of novel SNPs in offspring compared to the CT group. These findings suggest that multigenerational metabolic challenge can constrain genetic variability and induce genome instability, potentially mediated by transposable element activity rather than widespread changes in DNA methylation. This work highlights the importance of studying both genome and epigenome dynamics under realistic, multigenerational exposure scenarios and suggests that early metabolic challenges can have long-lasting impacts on genomic architecture and evolutionary potential.

Background: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related mortality, with an increasing incidence and dismal 10% survival at 5 years despite best available care. PDAC is characterized by low tumour cellularity, recurrent driver mutation alterations, and substantial inter and intra-tumoural heterogeneity. The influence of individual genetic features such as sex and ancestry on tumour heterogeneity remains poorly understood. Methods: We assembled a cohort of PDAC across five datasets for a total of 7,527 tumours. Measures of genomic instability were assessed across sex and ancestry on a global level (SNV density and percent genome altered by copy number events), and at a gene level for recurrent events. KRAS variant frequencies, ploidy and mRNA abundance were also evaluated. Ancestry was assigned using ADMIXTURE from germline genotypes where available, and patient reported otherwise. Results: We observed ancestry-specific mutation patterns in global point mutation density, with higher SNV burden in African PDAC tumours and lower SNV burden in Asian PDAC tumours as compared to European tumours. We also identified an ancestry bias in the driver gene, GNAS, in which driver SNVs in GNAS was detected more frequently in those of Asian ancestry, and less frequently in those of African ancestry. No sex- or ancestry-associations were found for KRAS, but individual KRAS variants showed a trend towards ancestry-related variation. Conclusions: These findings demonstrate that ancestry contributes to the genomic heterogeneity of PDAC, shaping both global and gene-specific mutation patterns. This work underscores the importance of including diverse ancestries in PDAC molecular profiling efforts which may inform the development of tailored molecular diagnostics and targeted therapies.

BackgroundUnderstanding the role of non-coding genomic variation in speciation remains a major challenge in evolutionary biology. Here, we investigated whether regulatory elements contribute to this process between Atlantic and Mediterranean lineages of European sea bass (Dicentrarchus labrax), a well-characterized case-study near speciation where barriers to introgression exist in the presence of connectivity between diverging populations. ResultsWe generated a novel, highly contiguous genome assembly, which was annotated at the epigenomic level using ATAC-seq and ChIP-seq with six embryonic developmental stages and five tissue types in adult fish, identifying thousands of promoters, enhancers, and open chromatin regions. Integrating this annotation with whole-genome sequence data from 65 individuals across three geographically distinct populations, we identified 57,505 outlier SNPs and 332 structural variants (SVs) showing elevated differentiation between Atlantic and East Mediterranean lineages. Outlier SVs affected key regulatory elements and coding genes, while outlier SNPs were enriched in regulatory elements, particularly enhancers active in adult tissues. Local genomic divergence correlated positively with regulatory element density, especially on chromosomes 1, 9, and 18, which are enriched in genes related to osmoregulation, immune response, and oxidative stress -- processes relevant to adaptation across contrasting marine environments. ConclusionsThese findings support a major role for regulatory variation in driving deep lineage divergence through local adaptation.

Pseudomonas aeruginosa (P. aeruginosa) is the primary pathogen responsible for morbidity and mortality in patients with cystic fibrosis (CF). Its genomic plasticity and constant selective pressure from antimicrobial treatments have favored the emergence of multidrug-resistant clones. This study conducted a comparative genomic analysis of 41 P. aeruginosa isolated from pediatric patients with CF in Mexico from 2015 to 2024, with the aim of characterizing their evolutionary dynamics, resistome, and virulome. Whole-genome sequencing (MGI, Illumina, and PacBio platforms) was used, with de novo assemblies performed using Unicycler v0.4.8 on the BV-BRC platform. The databases used for the resistome were CARD and NDARO, and for the virulome, VFDB. Phylogenetic reconstruction was based on core-genome alignments generated with Roary v3.13.0, with maximum likelihood reconstruction performed in IQ-TREE v2.1.2. The statistical significance of the segregation of resistance and virulence patterns was evaluated using PERMANOVA analysis. The results revealed a significant clonal prevalence of sequence types (ST) 307 and ST 167. Phylogenomic analysis grouped the isolates into three main clades; Clade 1 stood out for having the highest resistance gene load (mean of 75 genes/genome), establishing itself as the main reservoir of multidrug-resistant profiles. Genotype-phenotype concordance reached 65.5% overall, with high accuracy for aminoglycosides (87.8%) and fluoroquinolones (82.9%). Furthermore, virulome analysis identified 67 distinct patterns that were significantly segregated among the clades (PERMANOVA: R2=0.31, p=0.001). These findings demonstrate that the evolution of P. aeruginosa lineages in the pediatric clinical setting involves parallel and coordinated adaptations in both their resistance potential and their virulence arsenal. This study underscores the need to adopt a multidisciplinary approach to the clinical management of chronic P. aeruginosa infections in pediatric patients. The persistence of extensively drug-resistant (XDR) strains calls for the integration of genomic surveillance and functional diagnostics, as well as the search for therapeutic alternatives for the clinical management of patients with cystic fibrosis.

ABSTRACT Objective: Skeletal muscle growth rate differs substantially among diverse pig breeds. In the present study, RNA-seq was performed to identify differentially expressed genes (DEGs) across pig breeds with distinct growth rates. CCND2 showed obvious differential expression among pig breeds. Although CCND2 is essential for cell cycle control, its biological function in skeletal muscle differentiation has rarely been reported. This work aims to identify DEGs in pig breeds with different growth rates and explore CCND2 role in porcine skeletal muscle development. Methods: RNA-seq was performed to identify differentially expressed genes (DEGs) across pig breeds with distinct growth rates. Weighted gene co-expression network analysis (WGCNA) was used to identify gene modules potentially associated with skeletal muscle growth and development. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed to validate the role of CCND2 in skeletal muscle differentiation. Results We identified 5,416, 4,846, and 2,777 DEGs in the Duroc pig (DU) vs. Jiangquan black pig (JQ), DU vs. Laiwu pigs (LW), and JQ vs. LW comparison groups, respectively. WGCNA identified two gene modules (lightgreen and darkmagenta) potentially associated with skeletal muscle growth and development. CCND2 exhibited differential expression in comparisons between DU vs JQ and DU vs LW, with higher expression observed in Duroc pigs. qRT-PCR analysis revealed that overexpression of CCND2 at day 6 (D6) of cell differentiation significantly increased the expression levels of CCND2 gene and markers of myogenic differentiation (p < 0.05). Conversely, knockdown of CCND2 significantly decreased the expression levels of CCND2 gene and myogenic differentiation markers (p < 0.05). Conclusions This study identified breed-specific DEGs and key gene modules in porcine skeletal muscle and verified that CCND2 promotes myogenic differentiation. These findings provide a genetic theoretical basis for further investigations into skeletal muscle growth and development. Keywords pig, skeletal muscle, transcriptome, WGCNA, CCND2

Numerous genome-wide association studies in rice have identified loci associated with diverse agronomic traits. However, interpreting the regulatory and functional significance of these loci remains challenging because each locus often contains multiple variants in linkage disequilibrium, many of which lie in non-coding regions. Here, we present a method for prioritizing non-coding variants within an associated locus by integrating chromatin feature information predicted by a pretrained DNA language model fine-tuned on rice ChIP-seq and ATAC-seq datasets. We demonstrate the utility of our method through three case studies. In a post-GWAS analysis of heat tolerance, prioritized variants overlapped promoters of candidate genes previously identified through integrated GWAS and transcriptomic analyses, providing independent support for their potential regulatory roles. For the high-yield gene DEP1, promoter variant prioritization combined with in silico saturation mutagenesis identified a localized regulatory region enriched for high-impact mutations overlapping predicted transcription factor binding sites. For the drought-associated gene OsHAK1, the highest-ranked variant was predicted to be associated with chromatin features in a manner consistent with the reported co-occurrence of H2Bub with H3K4 methylation marks in plants. Overall, these results demonstrate the utility of our method for functionally informative prioritization of non-coding variants, facilitating the interpretation of GWAS loci and the identification of candidate regulatory variants in rice.

Environmental exposures can induce epigenetic modifications that persist across generations, potentially contributing to the transmission of environmentally-induced phenotypes. However, the extent to which such persistence of molecular changes are maintained independently of genetic mechanisms remains unclear. In this study, we investigated the evolution and genetic determinism of DNA methylation (DNAm) variation across three successive generations of Japanese quails (G0 to G2) belonging to two epilines, defined according to whether their female ancestor had received genistein supplementation (epi+) or not (epi-). Using reduced representation bisulfite sequencing (RRBS), we characterised CpG methylation patterns and identified differentially methylated cytosines (DMCs) and regions (DMRs) between control and genistein-supplemented epilines. Out of 112,745 CpG sites analysed, no DMCs or DMRs were detected between epilines in the first generation following exposure (G0), whereas 621 DMCs (36 DMRs) were identified in G1, and 1,381 DMCs (101 DMRs) in G2. Despite this progressive increase of differential methylation sites, mean differences in methylation rate between epilines were globally small. Similarly, genome-wide genetic differentiation between epilines was very limited. Overall, SNP-based heritability of DNA methylation was 0.19 indicating that genetic variation accounted for only a small part of the global variation of DNAm levels. By contrast, DNAm at differentially methylated CpG sites was extensively under genetic control, with a mean SNP-based heritability > 0.5, and meQTL analysis further identified significant associations between SNPs and methylation levels. Together, these results indicate that local DNAm differentiation in quail is under substantial genetic regulation, while the limited genetic differentiation between groups suggests that genetic variation alone can not fully explain the progressive accumulation of methylation differences following an ancestral exposure to genistein. These results support DNAm as a plausible molecular candidate mechanism in the multigenerational response to environmental exposure and highlight the complex interplay between genetic and epigenetic regulations in the inheritance of phenotypes.

The goat research community benefits from one of the most comprehensive livestock genome assemblies, ARS1. However, the rich diversity of goat breeds suggests that a single individual genome is not enough to represent the species full genomic content. Third-generation sequencing technologies have recently made it possible to generate very high quality assemblies that should enable to elucidate unique and/or complex phenotype determinisms depending on genetic structural variations. In the SeqOccin project, nine goat breeds were selected to represent French goat genetic diversity: Alpine, Boer, Creole, Fosses, Poitevine, Provencale, Pyreneenne, Rove and Saanen. High-molecular-weight DNA was extracted from blood samples and subjected to long read whole-genome sequencing. There reads were used to generate de novo assemblies and were aligned onto the public reference to call structural variants. PacBio HiFi and parental Illumina reads were used to assemble phased haplotypes of the Alpine breed. For the other breeds, PacBio raw (CLR) or ONT reads were generated. Our work demonstrates the feasibility of assembling genomes with a reasonable amount of data and appropriate software packages, delivering one high-quality assembly and 8 standard goat assemblies that pave the way for pangenome construction and structural variant functional studies.

The unicellular algae cell cycle can be divided into several phases, including the commitment point (CP), a point-of-no-return where the cell decides to divide, presumably based on reaching a critical cell size. Light plays a crucial role in the fitness of photosynthetic algal cells, affecting both CP timing and the number of daughter cells produced. So far, only few genes involved in CP have been described, and the presumed sizer and its signal(s) remain unidentified. Using synchronized cells and varying light intensities as a proxy, we explored the effects of light intensity in Chlamydomonas reinhardtii and observed both physiological and transcriptional changes occurring before and after CP under low light (LL) (100 {micro}mol m-{superscript 2} s-{superscript 1}) and optimal light (OL) (500 {micro}mol m-{superscript 2} s-{superscript 1}) conditions. Although CP was delayed by approximately 6 hours in LL, resulting in smaller mother cells and fewer daughter cells, the cells divided at the same time in both conditions. Overall, nucleic acid, protein, and energy reserve levels were lower in LL, with almost no starch produced. RNA-seq analysis identified several core genes shared between both conditions, with 201 genes expressed only in pre-CP1, 161 genes specific to post-CP1, and 582 shared across different phases. In LL, RNA-seq analysis showed an increase in differentially expressed genes (DEGs) in pre-CP1 compared to post-CP1, with an emphasis on photosynthesis, RNA metabolism, and organelle production before commitment, and cell division-related pathways (microtubules, DNA recombination) after CP1. In OL, the number of DEGs increased in post-CP1 by approximately 41% compared to pre-CP1, with a strong emphasis on protein production throughout the cell cycle.

Purpose: Autosomal Dominant Polycystic Kidney Disease is the most common monogenic kidney disease and largely due to variants in PKD1. We aimed to assess pathogenicity evidence for PKD1 missense variants in disease databases and evaluate in silico pathogenicity prediction tool performance. Methods: PKD1 missense variants reported as pathogenic, likely pathogenic or likely benign were extracted from ClinVar and PKDB. Variants were re-classified using ACMG/AMP criteria to identify "truth sets" of pathogenic and benign variants. In silico scores were obtained from five tools (SIFT, PolyPhen-2, CADD, REVEL, AlphaMissense) and evaluated using established thresholds. A Receiver Operating Characteristic curve analysis was performed using the PKD1 variant truth sets. Results: 346/389 (89%) reported disease-causing missense variants in PKD1 were downgraded to Variants of Unknown Significance (VUS) using current classification criteria. Based on current thresholds, REVEL achieved the highest sensitivity of 62%, with specificity of 79%. AlphaMissense was the only tool not to misclassify any truth set variants, but many of the variant scores were between the pathogenic and benign thresholds. Conclusion: A large majority of PKD1 missense variants are classified as VUS with current pathogenicity criteria. Commonly used in silico tools, applied with established genome-wide thresholds, do not reliably distinguish pathogenic and benign missense variants in PKD1.

The Enterobacteriaceae pathogens like E. coli, Shigella, Klebsiella and Salmonella cause a wide range of gastrointestinal and other mucosal infections when the contaminated food and water are consumed. Conventional microbiological methods are time-consuming and laborious process to detect these pathogens. Molecular assays are fast and sensitive to detect the pathogens. But a potential target for the detection is very essential to develop a molecular assay for the rapid detection. In this study, we explored the cpn60 UT region as a potential target for the detection of significant Enterobacteriaceae members like E. coli, Shigella sp, Salmonella, Klebsiella etc. We did the phylogenetic analysis of cpn60 UT region among the Enterobacteriaceae pathogenic groups. This analysis has revealed the presence of 13 clusters and two oligonucleotide regions with highest nucleotide conservation. Targeting these regions we have designed a set of oligonucleotide primer pairs and developed a SYBR green real time PCR assay to detect the Enterobacteriaceae pathogenic members. The degenerate oligonucleotide primers designed amplified a 132 bp UT region of cpn60 gene present in Enterobacteriaceae pathogens. The sensitivity of the assay was initially investigated by 10-fold dilution series of overnight broth culture of E. coli ATCC 10536 and had a detection limit of 101 CFU/mL. The specificity was assessed by comparing the amplification plot of a set of Enterobacteriaceae pathogens and non-Enterobacteriaceae members like Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. monocytogenes) and found to detect all the Enterobacteriaceae cultures included in the study whereas amplification was not seen in S. aureus and L. monocytogenes.

Vibrio harveyi is a primary causative agent of vibriosis, leading to catastrophic economic losses in the blackspotted croaker (Protonibea diacanthus) aquaculture. However, the genetic architecture underlying its resistance remains poorly understood. In this study, we conducted the first integrated genome-wide association study (GWAS) and genetic differentiation analysis (F_ST) to investigate the genetic basis of V. harveyi resistance. A total of 450 juveniles were challenged, and 300 individuals (comprising 150 resistant and 150 susceptible) were selected for whole-genome resequencing. Genomic heritability (h^2) was estimated at 0.374+/-0.29 for survival time (ST) and 0.237+/-0.185 for survival status (SS), indicating moderate genetic control. Notably, an exceptionally high genetic correlation (r_g = 0.999) was observed between ST and SS, while resistance traits were genetically independent of growth performance (r_g < 0.03). Through the convergence of GWAS and selection sweep signals, a major-effect QTL was identified on chromosome 4. Candidate gene prioritization identified high-confidence targets involved in post-transcriptional regulation and mRNA stability, specifically elavl4 (harboring a top-tier exon variant), rorb, cbpc6, and epn4. Functional enrichment analysis further revealed that resistance is significantly associated with "rRNA processing", "mRNA surveillance pathway", and "nucleocytoplasmic transport", suggesting that efficient RNA metabolic control is a critical determinant of host survival. These findings provide novel insights into the non-canonical immune mechanisms of P. diacanthus and offer robust molecular markers for marker-assisted selection and genomic selection, facilitating the development of disease-resistant strains in the sciaenid industry.

The sheep research community benefits from a genome assembly that has been refined over the last 15 years. However, the rich diversity of sheep breeds suggests that a single individual genome is not enough to represent the species full genomic content. Third-generation sequencing technologies have recently made it possible to generate high quality assemblies that should enable to elucidate unique and/or complex phenotype determinisms depending on genetic structural variations. In the SeqOccin project, twelve breeds were selected to represent (mostly French) sheep genetic diversity: Basco-Bearnaise, Black Belly, Blanche du Massif Central, Corse, Ile de France, Lacaune, Manech Tete Noire, Noire du Velay, Ouessant, Rambouillet, Vendeen, Soay. High-molecular-weight DNA was extracted from blood samples and subjected to long read whole-genome sequencing. The reads were used to generate de novo assemblies and were aligned onto the public reference to call structural variants. PacBio HiFi and parental Illumina reads were used to assemble phased haplotypes of the Lacaune breed. For the other breeds, PacBio raw (CLR) or ONT reads were generated. Our work demonstrates the feasibility of assembling genomes with a reasonable amount of data and appropriate software packages, delivering one high-quality assembly and 11 standard sheep assemblies that pave the way for pangenome construction and structural variant functional studies.

The black bean aphid, Aphis fabae is a crop pest and vector of insect-transmitted pathogens, comprising closely related sub-species with overlapping host ranges. In other Aphis species, over-expression of specific detoxification genes has been linked to insecticide tolerance. We present two chromosome-scale assemblies for a clonal A. fabae line, representing two phased haplotypes, generated using HiFi and Hi-C sequencing technologies. A comprehensive genome annotation, built with PacBio Iso-Seq data, was used to investigate genes underlying insecticide tolerance. Both genomes are comprised of four chromosomal blocks (haplotype 1: 427 Mb; haplotype 2: 396 Mb) with high BUSCO completeness (98.7%). Comparative genomics revealed an expansion of UDP-glycosyltransferases, whose expression is linked to insecticide detoxification in other Aphis species. These high-quality references provide a foundation for studying A. fabae sub-species and a genomic resource for investigating insecticide tolerance across the Aphis genus. Author summaryHere we have provided a comprehensive assembly and annotation for further study into the Black bean aphid, Aphis fabae, using up to date long-range sequencing technologies. The final assemblies for both haplotypes are chromosome length and consist of 4 main chromosome blocks, consistent with the literature. The A. fabae genome was found to contain an increase in copy number of UDP-glycosyltransferases, which have previously been linked to insecticide resistance. The work here will be a resource to those studying insecticide tolerance in crop pests, as well as the differences between A. fabae sub-species.

The genus Erwinia comprises a diverse group of bacteria associated with plants, insects, and the environment, including several economically important phytopathogens. The genus has been revised taxonomically many times, yet a thorough and genome-wide assessment of its evolutionary relationships and genomic diversity has been lacking. In this research, we carried out an extensive phylogenomic and comparative genomic analyses of the genus Erwinia using 104 genomes including historically important strains. Genome-wide analyses integrating average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), core-genome phylogenomics, pan-genome analysis, and comparative genomics resolved evolutionary relationships across the genus and identified multiple taxonomic inconsistencies. The pan-genome analysis revealed a relatively small core genome alongside an extensive accessory genome, underscoring the substantial genomic plasticity and ongoing diversification within the genus. The comparative analyses further showed pronounced lineage-specific variation in secretion systems, exopolysaccharide biosynthetic loci, flagellar gene clusters, genomic islands, prophages, and iron acquisition systems, suggesting that virulence-associated determinants have evolved through differential gene gain, loss, and conservation across distinct lineages, thereby facilitating host and ecological niche adaptation. This lineage-specific variation indicates that pathogenicity in the genus is not driven by a single conserved set of virulence determinants but instead reflects distinct combinations of virulence-associated genes. These findings refine the genomic framework of the genus Erwinia, provide evidence for taxonomic revision of several lineages, and improve our understanding of the evolutionary relationships, genomic diversification, and lineage-specific adaptations associated with host interactions and ecological specialization. Impact StatementThis study provides the first comprehensive genome-wide phylogenomic framework for the genus Erwinia, integrating taxonomy, pan-genome diversity, virulence-associated determinants, and mobile genetic elements across all 18 currently recognized species. Analyses resolve evolutionary relationships, uncover multiple taxonomic inconsistencies, identify previously unrecognized species-level lineages, including a putative novel Erwinia species PL328 isolated from Cornus florida (dogwood), and reveal lineage-specific genomic features. These findings establish a valuable genomic foundation for future studies of Erwinia evolution, taxonomy, and plant-microbe interactions. Data SummaryGenomes sequenced in this study were submitted to the NCBI database under the accession numbers: JCBCPT000000000

Translation initiation is a critical stage of protein synthesis in prokaryotes, where ribosomes assemble on mRNA to begin decoding genetic information. The interactions between ribosomal RNA (rRNA) and mRNA significantly influence this process, with features like ribosomal binding site (RBS) affecting ribosomal dynamics. However, direct experimental validation of these types of interaction is yet to be provided. In this study, we developed an optimized small sub-unit sequencing (SSU-seq) protocol for Escherichia coli, enabling the detailed examination of ribosomal complexes and subunits dynamics. Unlike Ribo-seq, which primarily maps fully assembled ribosomes, SSU-seq captures both ribosomal subunits and intact ribosomes, offering a comprehensive view of translation initiation and elongation events. By integrating experimental data with predictive models, our study advances the understanding of translation regulation in prokaryotes and underscores the utility of SSU-seq in decoding the complexities of ribosome-mRNA interactions. Our analysis reveals distinct patterns of small subunit distribution across the entire mRNAs with higher density near positions with stronger rRNA-mRNA hybridization. The results may suggest that rRNA-mRNA hybridization positions along the entire transcript facilitate efficient start codon recognition by maintaining proximity between the small subunit and the mRNA.

Antimicrobial resistance in Pseudomonas aeruginosa has emerged as a major clinical concern. Ciprofloxacin has potent intrinsic activity against P. aeruginosa, but resistance to this antibiotic is increasingly reported in clinical settings. In this study, P. aeruginosa ATCC 27853 strain was exposed to gradually increasing ciprofloxacin concentrations for 50 passages ([~]100 days). The final selected resistant strain (CIP-R) with 256-fold of minimum inhibitory concentration was studied further. Whole-genome sequencing revealed 12 genomic alterations, including known mutations in the quinolone resistance-determining genes gyrA (Thr83Ile), parC (Thr177Asn), parE (Glu459Lys). A duplication mutation in nfxB (Tyr153_Gly154dup) was also observed. In addition to these genes, we observed mutations in pilA, tadB, psdR, NP446_RS32055 (TRAP transporter permease), multiple dppA3 variants, and a prophage-associated hypothetical gene (NP446_RS24255), which have not been reported previously. The gyrA Thr83Ile substitution was conserved in 86.38% of ciprofloxacin-resistant clinical isolates retrieved from the NCBI database. Resistance acquisition was accompanied by slower growth, impaired swimming and swarming motility, diminished surface attachment, reduced biofilm formation. The resistant strain has enhanced {beta}-lactamase activity, and resistance to levofloxacin, cefepime, and meropenem together with sensitivity to piperacillin-tazobactam and aztreonam. This study highlights the gyrA Thr83Ile mutation as a key genomic marker for molecular screening of ciprofloxacin resistance and reveals secondary adaptive trade-offs that can be targeted for clinical diagnostic and therapeutic decision-making. In conclusion, achieving high-level ciprofloxacin resistance in P. aeruginosa involves non-target-site genomic adaptations and physiological trade-offs beyond classical target mutations.