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Abstract Staphylococcus aureus (S. aureus), particularly Methicillin-resistant S. aureus (MRSA), poses a significant global public health threat, necessitating advanced methodologies to enhance our understanding of this organism at the omics levels. This study introduces a refined protocol for constructing and curing high-density transposon mutant (tn-mutant) libraries in S. aureus, addressing the challenges associated with low transductant yields, and the complex genetic manipulation mechanism in Gram-positive bacteria. Our methodology employs a Himar1 transposon based on a two-plasmid system, leveraging Himar1’s high insertional efficiency in AT-rich organisms. Enhanced transduction efficiency was achieved through chloramphenicol pre-treatment and the use of modified enriched media. Complementing this, an optimized plasmid curing procedure ensured a representative and stable tn-mutant library. The protocol was successfully applied to multiple S. aureus strains, demonstrating an increase in mutant recovery and reduced post-curing impact. The method offers a robust approach for Transposon Insertion Sequencing (TIS) applications in S. aureus, enabling deeper insights into survival, resistance, and pathogenicity mechanisms. This protocol holds a significant potential for accelerating the construction of tn-mutant libraries in various S. aureus strains.

Ehrlichia muris subsp. eauclairensis is recognized as the etiological agent of human ehrlichiosis in Minnesota and Wisconsin. We describe the culture isolation of this organism from a field-collected tick and detail its relationship to other species of Ehrlichia. The isolate could be grown in a variety of cultured cell lines and was effectively transmitted between Ixodes scapularis ticks and rodents, with PCR and microscopy demonstrating a broad pattern of dissemination in arthropod and mammalian tissues. Conversely, Amblyomma americanum ticks were not susceptible to infection by the Ehrlichia. Histologic sections further revealed that the wild-type isolate was highly virulent for mice and hamsters, causing severe systemic disease that was frequently lethal. A Himar1 transposase system was used to create mCherryand mKate-expressing EmCRT mutants, which retained the ability to infect rodents and ticks. [ABSTRACT FROM AUTHOR]

Abstract 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 P BAD and antibiotic resistance cassettes to construct a series of derivative transposon vectors, p Mmch , p MKGR , p MCGR , p MXKGR , p MLKGR , p MSGR , and p MPR , based on the initial transposon p Mar2xT7. 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. [ABSTRACT FROM AUTHOR]

The gut microbiome may modulate intestinal immunity by luminal conversion of dietary amino acids to biologically active signals. The model probiotic organism Lactobacillus reuteri ATCC PTA 6475 is indigenous to the human microbiome, and converts the amino acid l-histidine to the biogenic amine, histamine. Histamine suppresses tumor necrosis factor (TNF) production by human myeloid cells and is a product of l-histidine decarboxylation, which is a proton-facilitated reaction. A transposon mutagenesis strategy was developed based on a single-plasmid nisin-inducible Himar1 transposase/transposon delivery system for L. reuteri. A highly conserved proton-chloride antiporter gene ( eriC), a gene widely present in the gut microbiome was discovered by Himar1 transposon (Tn)-mutagenesis presented in this study. Genetic inactivation of eriC by transposon insertion and genetic recombineering resulted in reduced ability of L. reuteri to inhibit TNF production by activated human myeloid cells, diminished histamine production by the bacteria and downregulated expression of histidine decarboxylase cluster genes compared to those of WT 6475. EriC belongs to a large family of ion transporters that includes chloride channels and proton-chloride antiporters and may facilitate the availability of protons for the decarboxylation reaction, resulting in histamine production by L. reuteri. This report leverages the tools of bacterial genetics for probiotic gene discovery. The findings highlight the widely conserved nature of ion transporters in bacteria and how ion transporters are coupled with amino acid decarboxylation and contribute to microbiome-mediated immunomodulation. [ABSTRACT FROM AUTHOR]

Methane is a key intermediate in the carbon cycle and biologically produced by methanogenic archaea. Most methanogens are able to conserve energy by reducing CO to methane using molecular hydrogen as electron donor (hydrogenotrophic methanogenesis), but several hydrogenotrophic methanogens can also use formate as electron donor for methanogenesis. Formate dehydrogenase (Fdh) oxidizes formate to CO and is involved in funneling reducing equivalents into the methanogenic pathway, but details on other factors relevant for formate-dependent physiology of methanogens are not available. To learn more about the factors involved in formate-dependent growth of Methanococcus maripaludis strain JJ, we used a recently developed system for random in vitro mutagenesis, which is based on a modified insect transposable element to create 2,865 chromosomal transposon mutants and screened them for impaired growth on formate. Of 12 M. maripaludis transposon-induced mutants exhibiting this phenotype, the transposon insertion sites in the chromosome were mapped. Among the genes, apparently affecting formate-dependent growth were those encoding archaeal transcription factor S, a regulator of ion transport, and carbon monoxide dehydrogenase/acetyl-CoA synthase. Interestingly, in seven of the mutants, transposons were localized in a 10.2 kb region where Fdh1, one of two Fdh isoforms in the organism, is encoded. Two transcription start sites within the 10.2 kb region could be mapped, and quantification of transcripts revealed that transposon insertion in this region diminished fdhA1 expression due to polar effects. [ABSTRACT FROM AUTHOR]

We report here the in vivo expression of the synthetic transposase gene himar1( a) in Streptomyces coelicolor M145 and Streptomyces albus. Using the synthetic himar1( a) gene adapted for Streptomyces codon usage, we showed random insertion of the transposon into the streptomycetes genome. The insertion frequency for the Himar1-derived minitransposons is nearly 100 % of transformed Streptomyces cells, and insertions are stably inherited in the absence of an antibiotic selection. The minitransposons contain different antibiotic resistance selection markers (apramycin, hygromycin, and spectinomycin), site-specific recombinase target sites ( rox and/or loxP), I- SceI meganuclease target sites, and an R6Kγ origin of replication for transposon rescue. We identified transposon insertion loci by random sequencing of more than 100 rescue plasmids. The majority of insertions were mapped to putative open-reading frames on the S. coelicolor M145 and S. albus chromosomes. These insertions included several new regulatory genes affecting S. coelicolor M145 growth and actinorhodin biosynthesis. [ABSTRACT FROM AUTHOR]

Mariner eukaryotic elements transpose randomly and independently of any host factors, making them ideal tools for random mutagenesis in bacteria, including genetically intractable microorganisms. The transposable element Himar1, a member of the mariner family of transposons, originally isolated from the horn fly ( Haematobia irritans), has thus been extensively used to generate large numbers of insertion mutants. Transposon-based approaches greatly facilitate studies of bacterial biology. We summarize the current mariner-based transposon tools and techniques for conducting genetic studies in bacteria. [ABSTRACT FROM AUTHOR]

Random transposon insertion libraries have proven invaluable in studying bacterial genomes. Libraries that approach saturation must be large, with multiple insertions per gene, making comprehensive genome-wide scanning difficult. To facilitate genome-scale study of the opportunistic human pathogen Pseudomonas aeruginosa strain PA14, we constructed a nonredundant library of PAl4 transposon mutants (the PA14NR Set) in which nonessential PAl4 genes are represented by a single transposon insertion chosen from a comprehensive library of insertion mutants. The parental library of PAl4 transposon insertion mutants was generated by using MAR2xT7, a transposon compatible with transposon-site hybridization and based on mariner. The transposon-site hybridization genetic footprinting feature broadens the utility of the library by allowing pooled MAR2xT7 mutants to be individually tracked under different experimental conditions. A public, internet-accessible database (the PA14 Transposon Insertion Mutant Database, http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/ home.cgi) was developed to facilitate construction, distribution, and use of the PA14NR Set. The usefulness of the PA14NR Set in genome-wide scanning for phenotypic mutants was validated in a screen for attachment to abiotic surfaces. Comparison of the genes disrupted in the PAl4 transposon insertion library with an independently constructed insertion library in P. aeruginosa strain PAO1 provides an estimate of the number of P. aeruginosa essential genes. [ABSTRACT FROM AUTHOR]

Streptococcus equi subsp. equi is the causative agent of the equine disease strangles. In this study we describe the development of an in vivo Himar1 transposon system for the random mutagenesis of S. equi and, potentially, other Gram-positive bacteria. We demonstrate efficient and random transposition of a modified mini-transposon onto the chromosome by Southern blot analysis and insertion site sequencing. Non-haemolytic mutants were isolated at a frequency of 0.2%, and acapsular mutants at a frequency of 0.04%. Taken together, these data demonstrate that in vivo Himar1 mutagenesis can be used for genomic-scale mutational analysis of S. equi, and is likely to be applicable to the study of other streptococci. [Copyright &y& Elsevier]

Vibrio vulnificus, a halophilic estuarine bacterium causing a rapidly progressing fatal septicemia, is highly cytotoxic to eukaryotic cells. To identify new virulence factors associated with cytotoxicity, we constructed a mariner-based transposon (Tn Himar1) library of the highly virulent clinical isolate MO6-24/O having a double mutation in the hemolysin and protease genes. The Himar1 mutant library was extensively screened for the mutants showing decreased cytotoxicity to HeLa cells. We selected a cytotoxicity defective mutant having a Himar1 insertion in an open reading frame showing 96% identity to Vibrio parahaemolyticus FlgC, a flagella basal body rod protein. The Tn Himar1 insertion mutation also resulted in a significant decrease in motility, adhesion, cytotoxicity, and lethality to mice. This is the first report showing that flg genes, which are components of the flagellum biogenesis gene cluster, might play an important role in the virulence of V. vulnificus. [Copyright &y& Elsevier]

Transposon sequencing (Tn-seq) has greatly accelerated the rate at which gene function can be profiled in microbial organisms. This technique has been applied to the study of the dental caries pathogen Streptococcus mutans where it has been used to generate large transposon mutant libraries. Coupled with high-throughput sequencing and bioinformatics tools, culture of these transposon mutant libraries has facilitated the identification of essential and conditional essential genes. In this chapter, we describe a procedure for performing Tn-seq studies in S. mutans that covers pooled transposon mutant construction, in vitro culture, and DNA library sequencing and data analysis., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Background Genetic systems have been developed for Chlamydia but the extremely low transformation frequency remains a significant bottleneck.  Our goal is to develop a self-replicating transposon delivery vector for C. trachomatis which can be expanded prior to transposase induction. Methods We made E. coli / C. trachomatis shuttle vectors bearing the Himar1 C9  transposase under control of the tet promoter and a novel rearrangement of the Himar1 transposon with the β-lactamase gene.  Activity of the transposase was monitored by immunoblot and by DNA sequencing. Results We constructed pSW2-mCh-C9, a C. trachomatis plasmid designed to act as a self-replicating vector carrying both the Himar1 C9  transposase under tet promoter control and its transposon.  However, we were unable to recover this plasmid in C. trachomatis following multiple attempts at transformation. Therefore, we assembled two new deletion plasmids pSW2-mCh-C9-ΔTpon carrying only the Himar1 C9  transposase (under tet promoter control) and a sister vector (same sequence backbone) pSW2-mCh-C9-ΔTpase carrying its cognate transposon.  We demonstrated that the biological components that make up both pSW2-mCh-C9-ΔTpon and pSW2-mCh-C9-ΔTpase are active in E. coli.  Both these plasmids could be independently recovered in C. trachomatis. We attempted to perform lateral gene transfer by transformation and mixed infection with C. trachomatis strains bearing pSW2-mCh-C9-ΔTpon and pSW2-RSGFP-Tpon (a green fluorescent version of pSW2-mCh-C9-ΔTpase).  Despite success in achieving mixed infections, it was not possible to recover progeny bearing both versions of these plasmids. Conclusions We have designed a self-replicating plasmid vector pSW2-mCh-C9 for C. trachomatis carrying the Himar1 C9  transposase under tet promoter control.  Whilst this can be transformed into E. coli it cannot be recovered in C. trachomatis.  Based on selected deletions and phenotypic analyses we conclude that low level expression from the tet inducible promoter is responsible for premature transposition and hence plasmid loss early on in the transformation process., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Skilton RJ et al.)

Generation of a random transposon mutant library is advantageous in Leptospira as site-directed mutagenesis remains a challenge, especially in pathogenic species. This procedure is typically completed by transformation of Leptospira with a Himar1 containing plasmid via conjugation with Escherichia coli as a donor cell. Here we describe the methodology to generate random transposon mutants in the saprophyte Leptospira biflexa via conjugation of plasmid pSW29T-TKS2 harbored in E. coli β2163. Determination of transposon insertion site by semi-random nested PCR will also be described. A similar methodology may be employed to generate Tn mutants of pathogenic Leptospira species.