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PubMed2026

Comparative essentialome analysis of six Pectobacteriaceae strains using the TNSEEK pipeline identifies conserved and strain-specific fitness determinants.

Transposon sequencing (Tn-seq) is a powerful technique for defining the essential genes required for bacterial survival. However, gene essentiality can vary significantly across taxonomic levels, and comparing large Tn-seq datasets from multiple strains presents considerable analytical challenges. To address this, we developed TNSEEK, a fully automated bioinformatics pipeline for the systematic and comparative analysis of Tn-seq experiments. We applied TNSEEK to analyse newly generated data for six soft rot Pectobacteriaceae strains, encompassing species from the Dickeya and Pectobacterium genera, grown in a rich medium. This approach identified a core essentialome of 225 genes, primarily involved in fundamental cellular maintenance, conserved across all 6 strains, a set comparable in size to that of the neighbouring Enterobacteriaceae family. Only a few genus-specific essential genes were found, highlighting interesting distinct metabolic capabilities between Dickeya and Pectobacterium genera. In striking contrast, we discovered a large variable essentialome comprising 181 strain-specific genes, many of which are of unknown function. A portion of these strain-specific essential genes are components of defence systems and prophage genomic regions. The unexpected essentiality of selected components of these modules is consistent with cellular dependency on cognate toxic, restriction or immunity functions encoded by defence-associated loci under the tested growth condition. Furthermore, a comparison with the Escherichia coli essentialome demonstrates that discrepancies in gene essentiality can often be attributed to differences in growth conditions, particularly temperature, as well as variations in genetic redundancy. In conclusion, the TNSEEK pipeline provides a reproducible framework for comparative analysis of mariner/Himar1 Tn-seq datasets across multiple strains.

Pectobacterium↗

PubMed2026

Applications of transposon-insertion sequencing for understanding bacterial physiology.

Transposon-insertion sequencing (Tn-seq) couples transposon mutagenesis with next-generation sequencing to identify the transposon insertion site for thousands of mutants in parallel. It is a powerful technology with a myriad of uses beyond the identification of essential genes required for a cell to grow and divide. Tn-seq is particularly useful as a high-throughput method to assign function to function-unknown genes, which have increased steadily with the abundance of newly sequenced bacterial genomes. Tn-seq has now been adapted for use in over 100 bacterial species. Here, we summarize the applications of Tn-seq for querying bacterial physiology and discuss some of the possible applications for the future.

DNA Transposable Elements↗

PubMed2026

Azotobacter vinelandii gene fitness following carbon shift from sucrose to acetate, succinate and glycerol.

Nitrogen-fixing microbes are a primary contributor of this important nutrient to the global nitrogen cycle. Biological nitrogen fixation (BNF) through the enzyme nitrogenase requires extensive energy that in whole cells is generally studied during the oxidation of carbohydrates such as sugars. The nitrogen-fixing bacterium Azotobacter vinelandii is a model diazotroph for the study of aerobic BNF. Much is known about metabolism in A. vinelandii when cultured on a simple medium where energy is provided primarily in the form of sucrose or glucose. Outside of the laboratory, this soil bacterium grows on metabolites primarily derived from plant root exudates or from the degradation of dead plant matter. In this work, we expand on previous studies looking at genes that are essential to BNF in A. vinelandii when grown on sucrose medium using transposon sequencing (Tn-seq). We applied Tn-seq to determine the genes essential to growth when the medium was shifted to acetate, succinate or glycerol as the primary carbon and energy source to fuel both growth and BNF. A global overview of the genes of central metabolism and those directing substrates toward central metabolism, along with a selection of unexpected genes that were essential for specific growth substrates, is provided.

Azotobacter vinelandii↗

PubMed2025

Landscape of essential growth and fluconazole-resistance genes in the human fungal pathogen Cryptococcus neoformans.

Fungi can cause devastating invasive infections, typically in immunocompromised patients. Treatment is complicated both by the evolutionary similarity between humans and fungi and by the frequent emergence of drug resistance. Studies in fungal pathogens have long been slowed by a lack of high-throughput tools and community resources that are common in model organisms. Here we demonstrate a high-throughput transposon mutagenesis and sequencing (TN-seq) system in Cryptococcus neoformans that enables genome-wide determination of gene essentiality. We employed a random forest machine learning approach to classify the C. neoformans genome as essential or nonessential, predicting 1,465 essential genes, including 302 that lack human orthologs. These genes are ideal targets for new antifungal drug development. TN-seq also enables genome-wide measurement of the fitness contribution of genes to phenotypes of interest. As proof of principle, we demonstrate the genome-wide contribution of genes to growth in fluconazole, a clinically used antifungal. We show a novel role for the well-studied RIM101 pathway in fluconazole susceptibility. We also show that insertions of transposons into the 5' upstream region can drive sensitization of essential genes, enabling screenlike assays of both essential and nonessential components of the genome. Using this approach, we demonstrate a role for mitochondrial function in fluconazole sensitivity, such that tuning down many essential mitochondrial genes via 5' insertions can drive resistance to fluconazole. Our assay system will be valuable in future studies of C. neoformans, particularly in examining the consequences of genotypic diversity.

Cryptococcus neoformans↗

PubMed2025

Cross-species phenotypic profiling uncovers functional determinants of bacterial cold shock adaptation.

Temperature shifts impose broad physiological stress, requiring precise and dynamic regulatory programs to restore cellular homeostasis. While the heat shock response is well characterized, the mechanisms underlying cold shock response (CSR) remain less understood. To identify genes critical for cold adaptation, we applied transposon sequencing (Tn-seq) to monitor mutant fitness across the full course of CSR and sustained low-temperature growth in two mesophilic bacteria, Escherichia coli and Bacillus subtilis. In B. subtilis, phenotypic profiling revealed a temporally structured program: membrane fluidity and cell wall remodeling were most critical in the early stage of CSR, whereas post-transcriptional regulation became essential during late-stage recovery to reprogram gene expression and restore growth. Cross-species comparison uncovered both conserved and species-specific mechanisms, with RNA metabolism and ribosome/translation regulators playing broad roles. Specifically, we identified a conserved synergy between two ribosomal RNA methyltransferases, RsmA and RsmH, in promoting cold adaptation. In B. subtilis, mutants lacking these enzymes exhibited significant delay in translation recovery following cold-induced global inhibition. Together, these findings provide a comparative, systems-level view of bacterial cold adaptation and establish a framework for exploring stress responses in pathogens and extremophiles.

Cell envelope↗

PubMed2023

Genome-wide screen in human plasma identifies multifaceted complement evasion of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is a leading cause of bacteremia with a high mortality rate. We recently reported that P. aeruginosa forms a persister-like sub-population of evaders in human plasma. Here, using a gain-of-function transposon sequencing (Tn-seq) screen in plasma, we identified and validated previously unknown factors affecting bacterial persistence in plasma. Among them, we identified a small periplasmic protein, named SrgA, whose expression leads to up to a 100-fold increase in resistance to killing. Additionally, mutants in pur and bio genes displayed higher tolerance and persistence, respectively. Analysis of several steps of the complement cascade and exposure to an outer-membrane-impermeable drug, nisin, suggested that the mutants impede membrane attack complex (MAC) activity per se. Electron microscopy combined with energy-dispersive X-ray spectroscopy (EDX) revealed the formation of polyphosphate (polyP) granules upon incubation in plasma of different size in purD and wild-type strains, implying the bacterial response to a stress signal. Indeed, inactivation of ppk genes encoding polyP-generating enzymes lead to significant elimination of persisting bacteria from plasma. Through this study, we shed light on a complex P. aeruginosa response to the plasma conditions and discovered the multifactorial origin of bacterial resilience to MAC-induced killing.

Humans↗

PubMed2026

Identification of a putative RocS homolog through phenotypic profiling of uncharacterized essential genes in Streptococcus mutans.

Genome-wide viability catalogs produced by transposon sequencing (Tn-seq) and CRISPR interference (CRISPRi) have successfully mapped the essential genome of Streptococcus mutans . In this study, we combined predictive bioinformatics, conditional CRISPRi transcriptional silencing, transmission electron microscopy, transcriptomics, and genetic suppressor screens to investigate nine poorly characterized essential genes in S. mutans . From this screen, phenotypic and genetic analyses identified SMU_393 as a functional homolog of the pneumococcal chromosome segregation factor, RocS. Depletion of SMU_393 resulted in abnormal cell widening, hypersensitivity to DNA damage, and a significant subpopulation of anucleate cells. These phenotypes were bypassed by a spontaneous surface-exposed missense mutation ( dnaA Q197E ) within the AAA+ ATPase domain of the replication initiator. Together, this study refines annotations within the S. mutans essential genome and provides genetic insights into streptococcal chromosome segregation and cell cycle control.

Journal Article↗

PubMed2026

Dual roles of genes required for intrinsic resistance to clarithromycin in evasion of killing by serum complement in Haemophilus influenzae.

Macrolide antibiotics are commonly prescribed to treat Haemophilus influenzae respiratory tract infections. Studies have primarily focused on emerging H. influenzae strains with acquired macrolide resistance, while the bacterium's intrinsic resistance to antibiotics has been underexamined. Here, we used a genome-wide approach of transposon insertion-site sequencing to screen an H. influenzae mutant library grown in sub-inhibitory doses of the macrolide antibiotic clarithromycin (CLR) to identify 33 genes involved in intrinsic CLR resistance. Almost half of these genes are also needed for survival in the mouse lung. We focused on candidate genes necessary for both intrinsic macrolide resistance and lung survival. Two of these genes affect the outer-membrane composition of H. influenzae, orfH and omp26. Deletions of these genes in Rd and nontypeable H. influenzae clinical isolates, Hi375 and NT127, conferred sensitivity to CLR and polymyxin B and increased membrane permeability to ethidium bromide (EtBr). The omp26 mutant was sensitive to killing by human serum. Deletions of orfH or omp26 in an acrR mutant strain overexpressing a multidrug efflux pump abrogated resistance of the acrR mutant to CLR and restored permeability to EtBr. Thus, deletion of these genes not only mitigates the effects of an acquired resistance mechanism but also remarkably overrides it. Complementation of these deletion mutations restored CLR resistance and decreased permeability to EtBr. Our results indicate that the subset of genes with dual roles in intrinsic resistance and host lung survival may provide potential novel combination antimicrobial therapeutic targets.

Animals↗