Transposon Insertion Mutant Library Research Articles

TRNA modification profiling reveals epitranscriptome regulatory networks in Pseudomonas aeruginosa.

Transfer RNA (tRNA) modifications have emerged as critical posttranscriptional regulators of gene expression affecting diverse biological and disease processes. While there is extensive knowledge about the enzymes installing the dozens of post-transcriptional tRNA modifications - the tRNA epitranscriptome - very little is known about how metabolic, signaling, and other networks integrate to regulate tRNA modification levels. Here we took a comprehensive first step at understanding epitranscriptome regulatory networks by developing a high-throughput tRNA isolation and mass spectrometry-based modification profiling platform and applying it to a Pseudomonas aeruginosa transposon insertion mutant library comprising 5,746 strains. Analysis of >200,000 tRNA modification data points validated the annotations of predicted tRNA modification genes, uncovered novel tRNA-modifying enzymes, and revealed tRNA modification regulatory networks in P. aeruginosa. Platform adaptation for RNA-seq library preparation would complement epitranscriptome studies, while application to human cell and mouse tissue demonstrates its utility for biomarker and drug discovery and development.

The secondary metabolite hydrogen cyanide protects Pseudomonas aeruginosa against sodium hypochlorite-induced oxidative stress.

The high pathogenicity of Pseudomonas aeruginosa is attributed to the production of many virulence factors and its resistance to several antimicrobials. Among them, sodium hypochlorite (NaOCl) is a widely used disinfectant due to its strong antimicrobial effect. However, bacteria develop many mechanisms to survive the damage caused by this agent. Therefore, this study aimed to identify novel mechanisms employed by P. aeruginosa to resist oxidative stress induced by the strong oxidizing agent NaOCl. We analyzed the growth of the P. aeruginosa mutants ΔkatA, ΔkatE, ΔahpC, ΔahpF, ΔmsrA at 1 μg/mL NaOCl, and showed that these known H2O2 resistance mechanisms are also important for the survival of P. aeruginosa under NaOCl stress. We then conducted a screening of the P. aeruginosa PA14 transposon insertion mutant library and identified 48 mutants with increased susceptibility toward NaOCl. Among them were 10 mutants with a disrupted nrdJa, bvlR, hcnA, orn, sucC, cysZ, nuoJ, PA4166, opmQ, or thiC gene, which also exhibited a significant growth defect in the presence of NaOCl. We focussed our follow-up experiments (i.e., growth analyzes and kill-kinetics) on mutants with defect in the synthesis of the secondary metabolite hydrogen cyanide (HCN). We showed that HCN produced by P. aeruginosa contributes to its resistance toward NaOCl as it acts as a scavenger molecule, quenching the toxic effects of NaOCl.

Multiplexed fitness profiling by RB-TnSeq elucidates pathways for lignin-related aromatic catabolism in Sphingobium sp. SYK-6

Bioconversion of lignin-related aromatic compounds relies on robust catabolic pathways in microbes. Sphingobium sp. SYK-6 (SYK-6) is a well-characterized aromatic catabolic organism that has served as a model for microbial lignin conversion, and its utility as a biocatalyst could potentially be further improved by genome-wide metabolic analyses. To this end, we generate a randomly barcoded transposon insertion mutant (RB-TnSeq) library to study gene function in SYK-6. The library is enriched under dozens of enrichment conditions to quantify gene fitness. Several known aromatic catabolic pathways are confirmed, and RB-TnSeq affords additional detail on the genome-wide effects of each enrichment condition. Selected genes are further examined in SYK-6 or Pseudomonas putida KT2440, leading to the identification of new gene functions. The findings from this study further elucidate the metabolism of SYK-6, while also providing targets for future metabolic engineering in this organism or other hosts for the biological valorization of lignin.

Streptococcal autolysin promotes dysfunction of swine tracheal epithelium by interacting with vimentin.

Streptococcus suis serotype 2 (SS2) is a major zoonotic pathogen resulting in manifestations as pneumonia and septic shock. The upper respiratory tract is typically thought to be the main colonization and entry site of SS2 in pigs, but the mechanism through which it penetrates the respiratory barrier is still unclear. In this study, a mutant with low invasive potential to swine tracheal epithelial cells (STECs) was screened from the TnYLB-1 transposon insertion mutant library of SS2, and the interrupted gene was identified as autolysin (atl). Compared to wild-type (WT) SS2, Δatl mutant exhibited lower ability to penetrate the tracheal epithelial barrier in a mouse model. Purified Atl also enhanced SS2 translocation across STEC monolayers in Transwell inserts. Furthermore, Atl redistributed the tight junctions (TJs) in STECs through myosin light chain kinase (MLCK) signaling, which led to increased barrier permeability. Using mass spectrometry, co-immunoprecipitation (co-IP), pull-down, bacterial two-hybrid and saturation binding experiments, we showed that Atl binds directly to vimentin. CRISPR/Cas9-targeted deletion of vimentin in STECs (VIM KO STECs) abrogated the capacity of SS2 to translocate across the monolayers, SS2-induced phosphorylation of myosin II regulatory light chain (MLC) and MLCK transcription, indicating that vimentin is indispensable for MLCK activation. Consistently, vimentin null mice were protected from SS2 infection and exhibited reduced tracheal and lung injury. Thus, MLCK-mediated epithelial barrier opening caused by the Atl-vimentin interaction is found to be likely the key mechanism by which SS2 penetrates the tracheal epithelium.

PurA facilitates Edwardsiella piscicida to escape NF-κB signaling activation

The host NF-κB signaling pathway plays critical role in defensing against bacterial infection. However, bacteria also evolve strategies to escape from host clearance. Edwardsiella piscicida is a threatening pathogen in aquaculture, while the molecular mechanism of E. piscicida in inhibiting NF-κB signaling remains largely unknown. Herein, using E. piscicida transposon insertion mutant library combined with a NF-κB luciferase reporter system, we identified forty-six genes of E. piscicida, which were involved in inhibiting the NF-κB signaling activation in vitro. Moreover, we further explored the top 10 significantly changed mutants through zebrafish larvae infection model and validated that six genes were involved in inhibiting NF-κB activation in vivo. Specifically, we identified the adenylosuccinate synthase mutated strain (ΔpurA) infection exhibited a robust activation of NF-κB signaling, along with higher expression of cxcl8a and cxcl8b to mediate the recruitment of neutrophils in vivo. Taken together, these results identified the key factors of E. piscicida in inhibiting NF-κB activation, which will contribute to better understanding the pathogenesis of this important pathogen.

Identification of the Genes of the Plant Pathogen Pseudomonas syringae MB03 Required for the Nematicidal Activity Against Caenorhabditis elegans Through an Integrated Approach.

Nematicidal potential of the common plant pathogen Pseudomonas syringae has been recently identified against Caenorhabditis elegans. The current study was designed to investigate the detailed genetic mechanism of the bacterial pathogenicity by applying comparative genomics, transcriptomics, mutant library screening, and protein expression. Results showed that P. syringae strain MB03 could kill C. elegans in the liquid assay by gut colonization. The genome of P. syringae MB03 was sequenced and comparative analysis including multi locus sequence typing, and genome-to-genome distance placed MB03 in phylogroup II of P. syringae. Furthermore, comparative genomics of MB03 with nematicidal strains of Pseudomonas aeruginosa (PAO1 and PA14) predicted 115 potential virulence factors in MB03. However, genes for previously reported nematicidal metabolites, such as phenazine, pyochelin, and pyrrolnitrin, were found absent in the MB03 genome. Transcriptomics analysis showed that the growth phase of the pathogen considerably affected the expression of virulence factors, as genes for the flagellum, glutamate ABC transporter, phoP/phoQ, fleS/fleR, type VI secretion system, and serralysin were highly up-regulated when stationary phase MB03 cells interacted with C. elegans. Additionally, screening of a transposon insertion mutant library led to the identification of other nematicidal genes such as acnA, gltP, oprD, and zapE. Finally, the nematicidal activity of selected proteins was confirmed by heterologous expression in Escherichia coli.

Transposon Insertion Mutagenesis in Hyperthermophilic Crenarchaeon Sulfolobus islandicus.

Transposon insertion mutagenesis is a forward genetic approach that has been widely utilized for genetic characterization of bacteria and single-celled eukaryotes, and its applications are being rapidly expanded into a few archaeal model organisms for gene function analysis. Previously, we developed a Tn5-based in vivo transposon insertion mutagenesis system in the hyperthermophilic crenarchaeon S. islandicucs M.16.4 and defined the essential gene set under laboratory growth conditions. In this chapter, we will mainly focus on presenting details regarding the generation of a near-saturating transposon insertion mutant library in this crenarchaeal model. We envision that the traditional transposon-based forward mutagenesis screening paired with next generation sequencing will greatly speed up the exploration of archaeal genomic features.

Genome-wide identification of probiotic Escherichia coli Nissle 1917 (EcN) fitness genes during adhesion to the intestinal epithelial cells Caco-2.

Escherichia coli Nissle 1917 (EcN) is an efficient probiotic strain extensively used worldwide because of its several health benefits. Adhesion to the intestinal cells is one of the prerequisites for a probiotic strain. To identify the genes essential for the adhesion of EcN on the intestinal cells, we utilized a quantitative genetic footprinting approach called transposon insertion sequencing (INSeq). A transposon insertion mutant library of EcN comprising of ~17,000 mutants was used to screen the adherence to the intestinal epithelial cells, Caco-2. The transposon insertion sites were identified from the input and output population by employing next-generation sequencing using the Ion torrent platform. Based on the relative abundance of reads in the input and output pools, we identified 113 candidate genes that are essential for the fitness of EcN during the adhesion and colonization on the Caco-2 cells. Functional categorization revealed that these fitness genes are associated with carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis, post-translational modification, stress response, motility and adhesion, and signal transduction. To further validate the genes identified in our INSeq analysis, we constructed individual knock-out mutants in five genes (cyclic di-GMP phosphodiesterase (gmp), hda, uidC, leuO, and hypothetical protein-coding gene). We investigated their ability to adhere to Caco-2 cells. Evaluation of these mutants showed reduced adhesion on Caco-2 cells, confirming their role in adhesion. Understanding the functions of these genes may provide novel insights into molecular regulation during colonization of probiotic bacteria to the intestinal cells, and useful to develop designer probiotic strains.

Bacterial factors required for Streptococcus pneumoniae coinfection with influenza A virus

BackgroundStreptococcus pneumoniae is a common cause of post-influenza secondary bacterial infection, which results in excessive morbidity and mortality. Although 13-valent pneumococcal conjugate vaccine (PCV13) vaccination programs have decreased the incidence of pneumococcal pneumonia, PCV13 failed to prevent serotype 3 pneumococcal disease as effectively as other vaccine serotypes. We aimed to investigate the mechanisms underlying the co-pathogenesis of influenza virus and serotype 3 pneumococci.MethodsWe carried out a genome-wide screening of a serotype 3 S. pneumoniae transposon insertion mutant library in a mouse model of coinfection with influenza A virus (IAV) to identify the bacterial factors required for this synergism.ResultsDirect, high-throughput sequencing of transposon insertion sites identified 24 genes required for both coinfection and bacterial infection alone. Targeted deletion of the putative aminotransferase (PA) gene decreased bacterial growth, which was restored by supplementation with methionine. The bacterial burden in a coinfection with the PA gene deletion mutant and IAV in the lung was lower than that in a coinfection with wild-type pneumococcus and IAV, but was significantly higher than that in an infection with the PA gene deletion mutant alone. These data suggest that IAV infection alters host metabolism to benefit pneumococcal fitness and confer higher susceptibility to pneumococcal infection. We further demonstrated that bacterial growth was increased by supplementation with methionine or IAV-infected mouse lung homogenates.ConclusionsThe data indicates that modulation of host metabolism during IAV infection may serve as a potential therapeutic intervention against secondary bacterial infections caused by serotype 3 pneumococci during IAV outbreaks in the future.

Competitive advantage of oxygen-tolerant bioanodes of Geobacter sulfurreducens in bioelectrochemical systems

Oxidative stress greatly limits current harvesting from anode biofilms in bioelectrochemical systems yet insufficient knowledge of the antioxidant responses of electricigens prevents optimization. Using Geobacter sulfurreducens PCA as a model electricigen, we demonstrated enhanced oxygen tolerance and reduced electron losses as the biofilms grew in height on the anode. To investigate the molecular basis of biofilm tolerance, we developed a genetic screening and isolated 11 oxygen-tolerant (oxt) strains from a library of transposon-insertion mutants. The aggregative properties of the oxt mutants promoted biofilm formation and oxygen tolerance. Yet, unlike the wild type, none of the mutants diverted respiratory electrons to oxygen. Most of the oxt mutations inactivated pathways for the detoxification of reactive oxygen species that could have triggered compensatory chronic responses to oxidative stress and inhibit aerobic respiration. One of the mutants (oxt10) also had a growth advantage with Fe(III) oxides and during the colonization of the anode electrode. The enhanced antioxidant response in this mutant reduced the system's start-up and promoted current harvesting from bioanodes even in the presence of oxygen. These results highlight a hitherto unknown role of oxidative stress responses in the stability and performance of current-harvesting biofilms of G. sulfurreducens and identify biological and engineering approaches to grow electroactive biofilms with the resilience needed for practical applications.

Peptide 1018 inhibits swarming and influences Anr-regulated gene expression downstream of the stringent stress response in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that causes considerable human morbidity and mortality, particularly in nosocomial infections and individuals with cystic fibrosis. P. aeruginosa can adapt to surface growth by undergoing swarming motility, a rapid multicellular movement that occurs on viscous soft surfaces with amino acids as a nitrogen source. Here we tested the small synthetic host defense peptide, innate defense regulator 1018, and found that it inhibited swarming motility at concentrations as low as 0.75 μg/ml, well below the MIC for strain PA14 planktonic cells (64 μg/ml). A screen of the PA14 transposon insertion mutant library revealed 29 mutants that were more tolerant to peptide 1018 during swarming, five of which demonstrated significantly greater swarming than the WT in the presence of peptide. Transcriptional analysis (RNA-Seq) of cells that were inoculated on swarming plates containing 1.0 μg/ml peptide revealed differential expression of 1,190 genes compared to cells swarming on plates without peptide. Furthermore, 1018 treatment distinctly altered the gene expression profile of cells when compared to that untreated cells in the centre of the swarm colonies. Peptide-treated cells exhibited changes in the expression of genes implicated in the stringent stress response including those regulated by anr, which is involved in anaerobic adaptation, indicative of a mechanism by which 1018 might inhibit swarming motility. Overall, this study illustrates potential mechanisms by which peptide 1018 inhibits swarming surface motility, an important bacterial adaptation associated with antibiotic resistance, virulence, and dissemination of P. aeruginosa.

Identification of Drug Resistance Determinants in a Clinical Isolate of Pseudomonas aeruginosa by High-Density Transposon Mutagenesis.

With the aim to identify potential new targets to restore antimicrobial susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa isolates, we generated a high-density transposon (Tn) insertion mutant library in an MDR P. aeruginosa bloodstream isolate (isolate ID40). The depletion of Tn insertion mutants upon exposure to cefepime or meropenem was measured in order to determine the common resistome for these clinically important antipseudomonal β-lactam antibiotics. The approach was validated by clean deletions of genes involved in peptidoglycan synthesis/recycling, such as the genes for the lytic transglycosylase MltG, the murein (Mur) endopeptidase MepM1, the MurNAc/GlcNAc kinase AmgK, and the uncharacterized protein YgfB, all of which were identified in our screen as playing a decisive role in survival after treatment with cefepime or meropenem. We found that the antibiotic resistance of P. aeruginosa can be overcome by targeting usually nonessential genes that turn essential in the presence of therapeutic concentrations of antibiotics. For all validated genes, we demonstrated that their deletion leads to the reduction of ampC expression, resulting in a significant decrease in β-lactamase activity, and consequently, these mutants partly or completely lost resistance against cephalosporins, carbapenems, and acylaminopenicillins. In summary, the determined resistome may comprise promising targets for the development of drugs that may be used to restore sensitivity to existing antibiotics, specifically in MDR strains of P. aeruginosa.

Identification and study of InV as an inverse autotransporter family representative in Edwardsiella piscicida

Invasins and intimins, members of virulence-related adhesin family which is involved in attachment and adherence to epithelial cells during infection, are found in various pathogens. These pathogens can attach to enterocytes and lead to the formation of a pedestal-like structure. Invasins and intimins belong to type Ve secretion systems, and the N-terminal β-barrel domain acts as a translocation pore to secrete the C-terminal passenger domain. However, the relationship between invasins/intimins and type III secretion system (T3SS) has been poorly studied. Based on the transposon insertion mutant library of Edwardsiella piscicida, we got a transposon insertion mutant with significant T3SS defect and identified the mutated gene ETAE_0323 (named inV later). This gene encoded a protein with 2359 amino acid residues and was predicted to be an invasin. To study the relationship between InV and T3SS, strains with N-terminus or C-terminus deleted InV fragments were made. However, none of them was able to copy the phenotype of the transposon insertion mutant previously identified. The localization of InV in ΔT3SS strain was not significantly different from WT, suggesting that the T3SS defect in the transposon insertion mutant was likely to be caused by polar effect. Nevertheless, depletion of inV still showed dramatic internalization and virulence defect in HeLa cell and zebrafish model, respectively, suggesting InV as a virulence related protein.

Identification of Regulatory Genes and Metabolic Processes Important for Alginate Biosynthesis in Azotobacter vinelandii by Screening of a Transposon Insertion Mutant Library.

Azotobacter vinelandii produces the biopolymer alginate, which has a wide range of industrial and pharmaceutical applications. A random transposon insertion mutant library was constructed from A. vinelandii ATCC12518Tc in order to identify genes and pathways affecting alginate biosynthesis, and about 4,000 mutant strains were screened for altered alginate production. One mutant, containing a mucA disruption, displayed an elevated alginate production level, and several mutants with decreased or abolished alginate production were identified. The regulatory proteins AlgW and AmrZ seem to be required for alginate production in A. vinelandii, similarly to Pseudomonas aeruginosa. An algB mutation did however not affect alginate yield in A. vinelandii although its P. aeruginosa homolog is needed for full alginate production. Inactivation of the fructose phosphoenolpyruvate phosphotransferase system protein FruA resulted in a mutant that did not produce alginate when cultivated in media containing various carbon sources, indicating that this system could have a role in regulation of alginate biosynthesis. Furthermore, impaired or abolished alginate production was observed for strains with disruptions of genes involved in peptidoglycan biosynthesis/recycling and biosynthesis of purines, isoprenoids, TCA cycle intermediates, and various vitamins, suggesting that sufficient access to some of these compounds is important for alginate production. This hypothesis was verified by showing that addition of thiamine, succinate or a mixture of lysine, methionine and diaminopimelate increases alginate yield in the non-mutagenized strain. These results might be used in development of optimized alginate production media or in genetic engineering of A. vinelandii strains for alginate bioproduction.

The putative bacterial oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) suppresses antibiotic resistance and pathogenicity in Pseudomonas aeruginosa

The putative bacterial oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) suppresses antibiotic resistance and pathogenicity in Pseudomonas aeruginosa

Identification of new components of the RipC-FtsEX cell separation pathway of Corynebacterineae.

Several important human pathogens are represented in the Corynebacterineae suborder, including Mycobacterium tuberculosis and Corynebacterium diphtheriae. These bacteria are surrounded by a multilayered cell envelope composed of a cytoplasmic membrane, a peptidoglycan (PG) cell wall, a second polysaccharide layer called the arabinogalactan (AG), and finally an outer membrane-like layer made of mycolic acids. Several anti-tuberculosis drugs target the biogenesis of this complex envelope, but their efficacy is declining due to resistance. New therapies are therefore needed to treat diseases caused by these organisms, and a better understanding of the mechanisms of envelope assembly should aid in their discovery. To this end, we generated the first high-density library of transposon insertion mutants in the model organism C. glutamicum. Transposon-sequencing was then used to define its essential gene set and identify loci that, when inactivated, confer hypersensitivity to ethambutol (EMB), a drug that targets AG biogenesis. Among the EMBs loci were genes encoding RipC and the FtsEX complex, a PG cleaving enzyme required for proper cell division and its predicted regulator, respectively. Inactivation of the conserved steAB genes (cgp_1603–1604) was also found to confer EMB hypersensitivity and cell division defects. A combination of quantitative microscopy, mutational analysis, and interaction studies indicate that SteA and SteB form a complex that localizes to the cytokinetic ring to promote cell separation by RipC-FtsEX and may coordinate its PG remodeling activity with the biogenesis of other envelope layers during cell division.

MarTrack: A versatile toolbox of mariner transposon derivatives used for functional genetic analysis of bacterial genomes

The mariner transposon family of Himar1 has been widely used for the random mutagenesis of bacteria to generate single insertions into the chromosome. Here, a versatile toolbox of mariner transposon derivatives was generated and applied to the functional genomics investigation of fish pathogen Edwardsiella piscicida. In this study, we combined the merits of the random mutagenesis of mariner transposon and common efficient reporter marker genes or regulatory elements, mCherry, gfp, luxAB, lacZ, sacBR, and PBAD and antibiotic resistance cassettes to construct a series of derivative transposon vectors, pMmch, pMKGR, pMCGR, pMXKGR, pMLKGR, pMSGR, and pMPR, based on the initial transposon pMar2xT7. The function and effectiveness of the modified transposons were verified by introducing them into E. piscicida EIB202. Based on the toolbox, a transposon insertion mutant library containing approximately 3.0 × 105 distinct mutants was constructed to explore the upstream regulators of esrB, the master regulator of the type III and type VI secretion systems (T3/T6SS) in E. piscicida. Following analysis by Con-ARTIST, ETAE_3474, annotated as fabR and involved in fatty acid metabolism, was screened out and identified as a novel regulator mediating T3SS and T6SS expression. In addition, the fabR mutants displayed critical virulence attenuation in turbot. Due to the broad-range host compatibility of mariner transposons, the newly built transposon toolbox can be applied for functional genomics studies in various bacteria.

The regulatory network of Vibrio parahaemolyticus type VI secretion system 1.

SummaryType VI secretion systems (T6SSs) are widespread, tightly regulated, protein delivery apparatuses used by Gram‐negative bacteria to outcompete their neighbours. The pathogen, Vibrio parahaemolyticus, encodes two T6SSs. These T6SSs are differentially regulated by external conditions. T6SS1, an antibacterial system predominantly found in pathogenic isolates, requires warm marine‐like conditions and surface sensing for activation. The regulatory network that governs this activation is not well understood. In this work, we devised a screening methodology that allows us to easily monitor the outcome of bacterial competitions and thus to identify mutants that are defective in T6SS1‐mediated bacterial killing. The methodology, termed Bacterial Competition Fluorescence (BaCoF), relies on detection of a fluorescent signal as an indicator of the survival and growth of a T6SS‐sensitive, GFP‐expressing prey that has been co‐cultured with mutants derived from a T6SS+ attacker of interest. Using BaCoF, we screened a random transposon insertion mutant library and identified genes required for V. parahaemolyticus T6SS1 activation, among them TfoY and Tmk. We used epistasis experiments to determine the relationships between the newly identified components and other regulators that were previously described. Thus, we present here a detailed biological understanding of the T6SS1 regulatory network.

Silencing of cyt-c4 led to decrease of biofilm formation in Aeromonas hydrophila.

Aquaculture suffers from a number of diseases caused by Aeromonas hydrophila. Biofilm can protect bacteria from antibiotic therapy. To identify the genes those play crucial roles in A. hydrophila biofilm formation, a library of mini-Tn10 transposon insertion mutants of A. hydrophila B11has been constructed, and 10 mutants were subjected to biofilm formation assay. The biofilm formation ability of mutant (B188) was significantly decreased compared with B11. The DNA sequence flanking the mini-Tn10 transposon inserted showed that an ORF of approximately 576 bp of the mutant strain B188 was inserted. This ORF putatively displays the highest identity (92%) with the cytochrome c4gene (cyt-c4) of A. hydrophila subsp. hydrophila ATCC 7966. Silencing cyt-c4 led to deficiencies in biofilm formation, adhesion, drug resistance and pathogenicity of A. hydrophila, which suggests that cyt-c4 plays crucial role in the biofilm formation and virulence mechanisms of A. hydrophila. ABBREVIATIONS: GEN: gentamycin; SDZ: sulfadiazine; AK: amikacin; P: penicillin; CFP: cefoperazone; LEV: levofloxacin; MH: minocycline; FFC: florfenicol; TE: tetracycline; AMP: ampicillin; KAN: kanamycin; STR: streptomycin; SXT: sulfamethoxazole/trimethoprim; DO: doxycycline; OT: Oxytetracycline.

RETRACTED: MarTrack: A versatile toolbox of mariner transposon derivatives used for functional genetic analysis of bacterial genomes

The mariner transposon family of Himar1 has been widely used for the random mutagenesis of bacteria to generate single insertions into the chromosome. Here, a versatile toolbox of mariner transposon derivatives was generated and applied to the functional genomics investigation of fish pathogen Edwardsiella piscicida. In this study, we combined the merits of the random mutagenesis of mariner transposon and common efficient reporter marker genes or regulatory elements, mcherry, gfp, luxAB, lacZ, sacBR, and PBAD and antibiotic resistance cassettes to construct a series of derivative transposon vectors, pMmch, pMKGR, pMCGR, pMXKGR, pMLKGR, pMSGR, and pMPR, based on the initial transposon pMar2xT7. The function and effectiveness of the modified transposons were verified by introducing them into E. piscicida EIB202. Based on the toolbox, a transposon insertion mutant library containing approximately 3.0 × 105 separated mutants was constructed to explore the upstream regulators of esrB, the master regulator of the type III and type VI secretion systems (T3/T6SS) in E. piscicida. Following analysis by Con-ARTIST, ETAE_2184 (renamed as EsrR) was screened out and identified as a novel regulator mediating T3SS expression. In addition, the esrR mutants displayed critical virulence attenuation. Due to the broad-range host compatibility of mariner transposons, the newly built transposon toolbox can be broadly applied for functional genomics studies in various bacteria.