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At least 19 recordsLinked to original sources
PubMed2005

Natural transposon mutagenesis of clinical isolates of Mycobacterium tuberculosis: how many genes does a pathogen need?

Transposable elements can affect an organism's fitness through the insertional inactivation of genes and can therefore be used to identify genes that are nonessential for growth in vitro or in animal models. However, these models may not adequately represent the genetic requirements during chains of human infection. We have therefore conducted a genome-wide survey of transposon mutations in Mycobacterium tuberculosis isolates from cases of human infection, identifying the precise, base-specific insertion sites of the naturally occurring transposable element IS6110. Of 294 distinct insertions mapped to the strain H37Rv genome, 180 were intragenic, affecting 100 open reading frames. The number of genes carrying IS6110 in clinical isolates, and hence apparently not essential for infection and transmission, is very much lower than the estimates of nonessential genes derived from in vitro studies. This suggests that most genes in M. tuberculosis play a significant role in human infection chains. IS6110 insertions were underrepresented in genes associated with virulence, information pathways, lipid metabolism, and membrane proteins but overrepresented in multicopy genes of the PPE family, genes of unknown function, and intergenic sequences. Population genomic analysis of isolates recovered from an organism's natural habitat is an important tool for determining the significance of genes or classes of genes in the natural biology of an organism.

Adolescent↗
PubMed2016

Design and synthesis of a minimal bacterial genome.

We used whole-genome design and complete chemical synthesis to minimize the 1079-kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.

Artificial Cells↗
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↗
PubMed2025

Antibiotic-resistant Acinetobacter baumannii can be killed by a combination of bacteriophages and complement.

Infections caused by multidrug-resistant Acinetobacter baumannii are an emerging global health threat. Although phages have shown promising results in treating bacterial infections, the mechanisms of the combined effect of phages and innate immunity on clearing A. baumannii remain unclear. Here, we report a synergistic effect of the complement system and phages on clearing multidrug-resistant A. baumannii. We show that A. baumannii rapidly adapts and becomes resistant to phage or serum complement by modifying the expression of capsule and lipooligosaccharides, which can be regulated through reversible transposon mutagenesis in the K locus. Compared to the encapsulated phenotype, the non-encapsulated, phage-resistant A. baumannii showed a higher level of membrane attack complex deposition and were susceptible to killing by complement. In contrast, the encapsulated phenotype escaped the complement system by shedding the membrane attack complex to the environment. Thus, while the complement system targets the non-encapsulated phenotype, the phage infects and eliminates the encapsulated subpopulation. These results suggest means of combatting antibiotic-resistant A. baumannii by a simultaneous treatment with phages and complement, a combination which can be supplemented further with antibacterial antibodies.

Acinetobacter baumannii↗
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

A rhamnose-rich O-antigen of Paraburkholderia phymatum MP20 is required for symbiosis with Mimosa pudica.

Paraburkholderia phymatum, a β-proteobacterium, forms a nitrogen-fixing symbiosis with many species of the large legume genus Mimosa as well as with common bean (Phaseolus vulgaris L.). Paraburkholderia are considered to have evolved nodulation independently from the well-studied α-proteobacteria symbionts of legumes. However, the detailed mechanisms important for β-rhizobia-legume symbiosis have not yet been determined. In this manuscript, we have sequenced the genome of P. phymatum MP20, a strain isolated from Mimosa pudica nodules, and utilized transposon mutagenesis to identify a mutant that showed delayed and ineffective nodulation of M. pudica. Further analysis revealed that the mutant strain produced an altered lipopolysaccharide lacking rhamnose containing O-antigen. Complementation with the wild-type gene restored the symbiosis. Microscopic analysis of the ineffective nodules showed that the mutant strain did not infect the cortical cells but was restricted to the endodermis. The results suggest that the O-antigen of P. phymatum is important for the bacterial infection of cortical cells and for nodule maturation. Further research will unveil the specific involvement of the glycosyltransferase gene in LPS biosynthesis and its impact on successful nodule formation by P. phymatum.IMPORTANCEThe nitrogen-fixing symbiosis between legumes and rhizobia is important for agricultural and environmental sustainability. The mechanisms of the symbiotic interactions are extensively studied using α-rhizobia. In contrast, mechanisms of symbiotic interactions important for β-rhizobia and their Caesalpinioid (mimosoid) legume hosts are not well known. Here, we describe the genome sequence of P. phymatum MP20, a β-rhizobia isolated from the nodules of M. pudica, and isolation and characterization of a transposon mutant defective in symbiosis. We demonstrate that the O-antigen of the LPS is required for nodulation and symbiotic nitrogen fixation. This study broadens our knowledge of symbiotic interactions in β-rhizobia and will lead to a better understanding of the wider rhizobial-legume symbiosis apart from the α-rhizobia.

Symbiosis↗
PubMed2026

Auditing bacterial dark-gene screens for superimposed open reading frame artefacts: A multi-layer analysis of Rv2438A in Mycobacterium tuberculosis.

Essentiality and knockdown-vulnerability screens can promote spurious bacterial open reading frames when those frames overlap essential genes, because such a frame inherits its neighbour's signals undiluted and therefore satisfies the screen's criteria better than a genuine small gene. We present a multi-layer audit that tests this failure mode across genome annotation, transposon mutagenesis, CRISPR interference, homology, transcript mapping, proteomics, and population variation. We apply it to Rv2438A, a 92-codon conserved hypothetical open reading frame of Mycobacterium tuberculosis ranked first by our own dark-gene target screen. Rv2438A is superimposed on the essential NAD synthetase locus nadE: 44% lies within its coding sequence on the opposite strand, and the remainder covers its promoter and transcription start site. Consequently, three of five Himar1 sites lie within nadE, no CRISPRi guide can target Rv2438A without binding nadE, and the cross-species hit maps to the same nadE start junction. Rv2438A lacks its own transcription start site and is absent from every proteomic dataset that detects nadE. A genome-wide scan identifies six short, overlapping, uncharacterised loci among 3907 annotated genes, but only Rv2438A combines overlap and essentiality with non-detection across all proteomic datasets; rare genome-wide, it ranked first among screen hits. We provide an implementable audit workflow and a codon-position control, but measure the control's sensitivity as only two of five genes with attested protein, limiting it to confirmatory use. Overlap coordinates and neighbour-specific experimental resolution should therefore be reported before bacterial dark genes are prioritised.

CRISPR interference↗
PubMed2024

Salmonella Typhimurium screen identifies shifts in mixed-acid fermentation during gut colonization.

How enteric pathogens adapt their metabolism to a dynamic gut environment is not yet fully understood. To investigate how Salmonella enterica Typhimurium (S.Tm) colonizes the gut, we conducted an in vivo transposon mutagenesis screen in a gnotobiotic mouse model. Our data implicate mixed-acid fermentation in efficient gut-luminal growth and energy conservation throughout infection. During initial growth, the pathogen utilizes acetate fermentation and fumarate respiration. After the onset of gut inflammation, hexoses appear to become limiting, as indicated by carbohydrate analytics and the increased need for gluconeogenesis. In response, S.Tm adapts by ramping up ethanol fermentation for redox balancing and supplying the TCA cycle with α-ketoglutarate for additional energy. Our findings illustrate how S.Tm flexibly adapts mixed fermentation and its use of the TCA cycle to thrive in the changing gut environment. Similar metabolic wiring in other pathogenic Enterobacteriaceae may suggest a broadly conserved mechanism for gut colonization.

Animals↗
PubMed2026

The AAA+ chaperone ClpB contributes to stress tolerance and pathogenesis in Mycoplasma bovis.

ClpB, an ATP-dependent molecular chaperone belonging to the Hsp100/Clp subfamily of AAA+ ATPases, plays a crucial role in protein disaggregation, thereby enhancing bacterial survival under stress conditions. Despite its well-conserved function in prokaryotes, the specific contributions of ClpB to the pathogenesis of the ruminant pathogen Mycoplasma bovis remain largely unexplored. In this study, we identified and functionally characterized a ClpB homolog in M. bovis. Biochemical assays confirmed that the recombinant ClpB protein exhibits intrinsic ATPase activity and, in cooperation with the DnaK chaperone system, efficiently mediates protein disaggregation in vitro. Through genome-wide transposon mutagenesis of the M. bovis HB0801 strain, we generated ClpB-deficient mutants that maintained normal growth kinetics and morphology at 37 °C but exhibited significant growth defects under thermal and oxidative stress conditions. Phenotypic analysis demonstrated that ClpB disruption attenuated key virulence traits, including impaired adhesion to host cells, marked reduction in biofilm formation, diminished pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) expression in BoMac cells. Furthermore, the reduced virulence of the ClpB mutant was investigated by DIA proteomic analyses, which revealed that the ClpB mutant strain altered distinct protein expression patterns related to proteostasis, including phosphotransferase system, serine-type peptidase activity, serine hydrolase activity, and chaperone-mediated protein folding that contribute to the stress response and virulence. These findings collectively demonstrate that ClpB serves as a multifunctional virulence determinant in M. bovis, orchestrating stress adaptation, host-pathogen interactions, and pathogenic potential through modulation of both protein quality control systems and virulence-associated pathways.

Mycoplasma bovis↗
PubMed2025

Common food preservatives induce an oxidative stress response in Salmonella enterica serovar Typhimurium.

Despite their frequent use, the mechanisms of action of common food preservatives are poorly understood. As there is a drive to develop alternative preservatives, understanding the mechanisms of action of current preservatives can inform the development of novel food preservatives to ensure their efficacy. Here, we used TraDIS-Xpress, a large-scale, genome-wide unbiased screen to determine the mechanisms of action of common food preservatives by determining the genes that affect preservative susceptibility in Salmonella enterica serovar Typhimurium. We identified genes associated with central metabolism and oxidative stress responses that were important for all four preservatives. Formate dehydrogenase activity and synthesis was crucial for survival in the presence of both sodium chloride and potassium chloride. We found some preservative-specific effects on pathogen susceptibility, for example, LPS synthesis which improved survival upon exposure to sodium nitrite but harmed survival when exposed to sodium chloride or potassium chloride. This research expands our understanding of how some current preservatives act and can inform the effective use of preservatives in current and emerging food products to ensure high standards of food safety.

Salmonella typhimurium↗
PubMed2025

The Streptococcus pyogenes mannose phosphotransferase system (Man-PTS) influences antimicrobial activity and niche-specific nasopharyngeal infection.

Streptococcus pyogenes is a human-adapted pathogen that can cause multiple diseases, including pharyngitis and skin infections. Although this bacterium produces many virulence factors, how S. pyogenes competes with the host microbiota is not well understood. Here, we detected antimicrobial activity from S. pyogenes MGAS8232 that prevented the growth of Micrococcus luteus. This activity was produced when cells were grown in 5% CO2 in M17 media supplemented with galactose; however, the addition of alternative sugars coupled with genome sequencing experiments revealed that the antimicrobial phenotype was not related to classical bacteriocins. To further determine genes involved in the production of this activity, a transposon mutant library in S. pyogenes MGAS8232 identified the mannose phosphotransferase system (Man-PTS), a major sugar transporter, as important for the antimicrobial phenotype. Loss-of-function transposon mutants linked to the antimicrobial activity were identified to also be involved in alternative sugar utilization, and additionally, the Man-PTS was further identified from an inadvertent secondary mutation in a bacteriocin operon mutant. Sugar utilization in the Man-PTS mutants demonstrated that galactose, mannose, and N-acetylglucosamine utilization was impaired. RNA-seq experiments in high and low glucose concentrations further characterized the Man-PTS as a glucose transporter; however, transcriptional regulators or virulence factors were not affected with the loss of the Man-PTS. Deletion of Man-PTS demonstrated defects in a mouse model of nasopharyngeal infection but not skin infection. This work suggests that the ability of S. pyogenes to utilize alternative sugars presented by glycans may play a role in acute infection and interactions with the endogenous microbial population existing in the nasopharynx.IMPORTANCEStreptococcus pyogenes is responsible for over 500,000 deaths per year primarily due to invasive infections and post-infection sequelae, although the most common manifestations include pharyngitis and impetigo. S. pyogenes can adapt to its environment through alternative sugar metabolism. Here, we identified an antimicrobial phenotype that was not bacteriocin-related but a by-product of alternative sugar metabolism. The mannose phosphotransferase system was involved in the production of the antimicrobial and was also important for S. pyogenes to utilize alternative sugars and establish nasopharyngeal infection but not skin infection. Overall, this study identified potential strategies used by S. pyogenes for interactions with the endogenous microbiota and further elucidated the importance of sugar metabolism in acute upper respiratory tract infection.

Streptococcus pyogenes↗
PubMed2026

A genetic manipulation tool based on the GP35 recombinase for targeted gene editing in mycoplasmas of ruminants.

Pathogenic ruminant mycoplasmas are major etiological agents in cattle and small ruminants and are responsible for substantial economic losses in the livestock industry. Progress in pathogenesis research and vaccine development has been hampered by a lack of effective genetic tools. The applicability of common genome editing platforms, such as CRISPR, is inherently restricted in these organisms owing to their minimal genomes, the absence of a cell wall, and low homologous recombination efficiency. Although transposon-mediated random mutagenesis and single-base editing are currently used in the editing of bovine mycoplasma, the stochastic nature of transposons, the risk of single-base random deamination, and limitations in editing window selection hinder the genetic manipulation of bovine mycoplasma. Here, we introduce a plasmid-based methodology that employs the GP35 recombinase from bacteriophage SPP1 to mediate long single-stranded DNA (ssDNA) recombineering, thereby enabling precise gene insertions and deletions in Mycoplasma bovis, with a positive-editing rate of 77.78% - 100%. This targeted system eliminates the risk of random deamination. Leveraging this tool, we generated a panel of M. bovis mutants affecting metabolic and virulence genes and obtained key insights into Mb0564, identified as a novel adhesin. The 192 to 287 aa region of GP35 is critical for interaction with SSB. Structural conservation analysis further suggested that this GP35-ssDNA editing system possesses a high potential for translation to other ruminant pathogens. Collectively, our approach expands the existing genetic toolkit for M. bovis, advances synthetic biology and M. bovis pathobiology, facilitates vaccine development, and strengthens the control of high-impact livestock diseases in line with the One Health framework.

Animals↗
PubMed2025

A platform supporting generation and isolation of random transposon mutants in Chlamydia trachomatis.

Chlamydia species represent a paradigm for understanding successful obligate intracellular parasitism. Despite limited genetic malleability, development of genetic tools has facilitated the elucidation of molecular mechanisms governing infectivity. Random mutagenesis approaches provide one of the most powerful strategies available to accomplish untargeted elucidation of gene function. Unfortunately, initial progress in transposon-mediated mutagenesis of Chlamydia has been challenging. To increase efficiency, we developed a plasmid-based system that couples conditional plasmid maintenance with a previously described strategy leveraging inducible expression of the Himar1-derived C9 transposase. Our pOri-Tn(Q) construct was maintained in Chlamydia trachomatis cultivated with antibiotics but was rapidly cured in the absence of antibiotic selection. pOri-Tn(Q) supported transposition events when transposase expression was induced during infection. Induction was accompanied by loss of the plasmid backbone when penicillin G was used to select for only the transposable element. C9 induction during iterative passaging was used to increase the overall insertion frequency and accumulate an expanded pool of transposon mutants. The approach supported isolation of individual mutant strains from the mixed pool, and whole-genome sequencing confirmed that the recovered strains harbored single insertions.IMPORTANCEChlamydia trachomatis is a prevalent human pathogen exerting a tremendous negative impact on human health. A complete understanding of how these bacteria create and maintain an intracellular niche and avoid/subvert host defense mechanisms to cause disease is lacking. The utility of transposon-mediated, random mutagenesis in supporting forward genetic studies is well established in a multitude of genetically tractable systems. This study reports the development of a plasmid-based system capable of generating mutant pools and supporting subsequent isolation of individual transposon mutants. This step is an important advance in providing a mechanism capable of supporting downstream studies interrogating chlamydial biology.

Chlamydia trachomatis↗
PubMed1979

Replication and gene functions of the bacteriocinogenic plasmid CloDF13.

The replication and genetic constitution of plasmid CloDF13 was studied using mutants of CloDF13 obtained by NTG mutagenesis, insertion of the ampicillin transposon Tn901, or deletion of particular CloDF13 DNA regions. Analysis of the polypeptides encoded by these mutant plasmids enabled us to locate six genes on the CloDF13 physical map. These genes cover about 60% of the coding capacity of CloDF13. A large part of the CloDF13 genome (about 30%) is involved in the conjugal transfer of this plasmid. This transfer region codes for at least two polypeptides, polypeptide B (61,000 daltons) and C (24,000 daltons). Those CloDF13 DNA regions responsible for the synthesis of the cloacin protein and immunity protein were also mapped on the plasmid genome. In addition we were able, using a copy mutant of CloDF13, CloDF13-cop3, to locate those DNA sequences involved in interaction with male-specific RNA phages and bacteriophage P1. For replication of CloDF13, two regions are essential. One region, from 43% to 64%, affects the stability of CloDF13-cop3 plasmids. In the case of the CloDF13-cop3 mutant, deletion of this region results in the generation of multimeric plasmid molecules accompanied by an impaired segregation of plasmid DNA molecules to daughter cells. The second region, from about 1.8% to 11.5%, contains an origin of replication as well as well as DNA sequences involved in the control of CloDF13 replication. The replication of CloDF13 starts at about 3% on the CloDF13 physical map and proceeds unidirectionally counter-clockwise.

Bacterial Proteins↗
PubMed1979

Plasmid cistrons controlling synthesis and excretion of the exotoxin alpha-haemolysin of Escherichia coli.

The synthesis and secretion of the toxic exoprotein alpha-haemolysin of E. coli PM152 is coded by the transmissible plasmid pHly152 (41 x 10(6) dalton) as shown by the transformation of the plasmid DNA and the isolation of mutants that are specifically altered in the synthesis and transport of haemolysin. These mutants were obtained by chemical mutagenesis and insertion of the ampicillin transposon (Tn3) into pHly152. Tn3 transposition was also used for the identification and the location of the cistrons on pHly152 essential for haemolysis. The EcoRI and HindIII fragments of the haemolytic plasmid pHly152 were cloned and used for the complementation of the haemolysis negative Tn3 insertion mutants. A DNA segment of 3.2 x 10(6) dalton could be thus identified which consists of at least three clustered cistrons necessary for haemolysis. Two of these cistrons are required for the formation of active haemolysin. At least one other cistron seems to be involved in the secretion of active haemolysin through the outer membrane of E. coli. The gene products determined by these cistrons were identified in minicells of E. coli. Their molecular properties were determined and their possible function in the formation and secretion of haemolysin will be discussed.

Escherichia coli↗
PubMed2025

Comprehensive profiling of activity and specificity of RNA-guided transposons reveals opportunities to engineer improved variants.

Recently discovered CRISPR-associated transposons (CASTs) are natural RNA-guided DNA transposition systems capable of single-step genomic integration of large DNA cargo. Wild-type CASTs exhibit low integration activity in heterologous systems; therefore, engineering efforts are required to develop therapeutically relevant tools. Here we developed a high-throughput dual genetic screen capable of accurately quantifying the relative activity and specificity of a large pool of CAST variants. Under the conditions of our screen, we discovered that the wild-type V-K CAST system can consistently achieve between 88% and 95% on-site targeting specificity. We used site-saturation mutagenesis of the conserved core transposition machinery (TnsB, TnsC, and TniQ) to reveal novel mechanistic insights into the function of these transposon proteins. Furthermore, we found that different components have varying trade-offs between activity and specificity, a critical aspect overlooked in conventional screening pipelines. These findings provide clear engineering principles for further optimization of CASTs. Finally, we identified several mutations that, together, enhance CAST activity up to four-fold while minimally impacting targeting specificity. These methods are a powerful tool to characterize the sequence-function landscape across multiple functional parameters while also providing a robust platform for developing enhanced genome-editing tools.

DNA Transposable Elements↗