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Vector Technology of Relevance to Nitrogen Fixation Research*
Published in Peter M. Gresshoff, Molecular Biology of Symbiotic Nitrogen Fixation, 2018
Reinhard Simon, Ursula B. Priefer
The principle of site-directed transposon mutagenesis can be briefly summarized as follows (Figure 4): a cloned DNA fragment is mutagenized by transposon insertion in E. coli, using one of the well-established standard procedures, e.g., with a λ::Tn5 vector or with a chromosomally located transposon.9,118 The insertion site can easily be mapped by restriction fragment analysis of the isolated plasmid DNA. The mutagenized DNA fragment is transferred back to its original host, where sequence homology allows recombination on both sides of the transposon insertion. This double crossover finally leads to the exchange of the wild-type gene for the transposon-mutated one. This technique not only enables the insertion of a transposon at a predetermined site in a target cell, but also allows very accurate analyses of genes and operons by comparing the physical map with the phenotype resulting from the gene replacement.
Proteus
Published in Dongyou Liu, Laboratory Models for Foodborne Infections, 2017
Paola Scavone, Victoria Iribarnegaray, Pablo Zunino
A traditional tool that allows for the identification of gene functions is random transposon mutagenesis. The basis of this technique is in the generation of mutant libraries that harbor a transposon insert, which abolishes the function of the affected gene. Transposons are mobile genetic elements that can move within the genome and can affect the function of gene expression. One of the limitations in identification and separation of nonvirulent mutants from a pool of mutants is that it is time consuming. In P. mirabilis, there are many studies that report the use of this technique. Recently, Holling et al. [55] used random transposon mutagenesis to identify genes involved in biofilm formation. They used a mini-Tn5Km2 transposon that was introduced into the wild-type P. mirabilis strain B4 by conjugal transfer from the donor organism, Escherichia coli S17.1λpir (Table 24.1). The mutants were screened by the crystal violet microtiter plate assay to identify bacterial impairment in catheter blockage. The end adjacent to the transposon was sequenced in order to identify the mutated gene.
How to discover new antibiotic resistance genes?
Published in Expert Review of Molecular Diagnostics, 2019
Linda Hadjadj, Sophie Alexandra Baron, Seydina M. Diene, Jean-Marc Rolain
Nowadays, faced with an unusual phenotype of AR, WGS is indispensable. Resistome analysis softwares are more upgraded, flexible and easier to use to understand most of the resistance phenotypes. If not, biochemical tests can define the nature of an AR mechanism and its enzymatic activity. In the presence of a gene conferring resistance, the functional genomic approach is a solid and powerful method. Regarding the resistance due to mutation of an existing gene, the functional genomic approach is inefficient, whereas transposon mutagenesis is the most adapted technique. The transposon mutagenesis can shed light on the role of certain genes involved in the mechanisms of AR. Once identified, these genes can be analyzed in order to find unusual mutations or deletions (Figure 5). Combination of biology and bioinformatic is primordial, especially for metagenomic studies for which the biological validation of new ARG is indispensable. The vulgarization of gene synthesis companies makes it possible to test bioinformatic predictions.
Transposon mutagenesis in oral streptococcus
Published in Journal of Oral Microbiology, 2022
Yixin Zhang, Zhengyi Li, Xin Xu, Xian Peng
Transposon mutagenesis is an effective forward genetic strategy for studying gene function by observing the phenotypic changes in mutated genes. Random mutants in a variety of prokaryotes have been created by using different transposon genes such as Tn3 derivatives, IS (insertion sequence) elements, Tn7, Tn5, and mariner. Since the advent of genome sequencing, techniques such as genetic footprinting, signature-tagged Mutagenesis (STM), transposon site hybridization (TraSH), and scanning Linker mutagenesis (SLM) have been developed [17]. And with the advent of next-generation sequencing (NGS), transposon insertion sequencing (TIS) combines it with large-scale transposon insertion mutations to evaluate the essentiality of genetic features and fitness contribution in the bacterial genome in the saturated random mutant libraries. The four TIS techniques published in 2009 include insertion sequencing (INSeq) in Bacteroides thetaiotaomicron [18], high-throughput insertion tracking by deep sequencing (HITS) in Haemophilus influenzae [19], transposon sequencing (Tn-Seq) in S. pneumoniae [20], and transposon-directed insertion site sequencing (TraDIS) in S. Typhi [21]. Those techniques have been widely used in various bacteria to study fitness and virulence, including Enterococcus faecalis [22], Vibrio parahaemolyticus [23], Salmonella enteritidis [24], Edwardsiella piscicida [25], Ralstonia solanacearum [26] and Pantoea [27]. Ultimately, TIS is a key tool for interpreting the rapidly increasing amount of genome sequencing data and is expected to shed light on the function of individual genome features. With the development of transposon technology, TIS has been reviewed from the perspectives of design and analysis [28,29]. Cain et al. discussed recent applications of TIS in answering general biological questions [30]. The present review focuses on oral microorganisms and highlights the application of transposon mutagenesis, including TIS, to oral streptococci, as well as research progress, aiming to better understand the relationship between oral streptococcal phenotype and genotype, which can help clarify the processes of colonization, virulence, and persistence and provides a more reliable basis for investigating relationships with host ecology and disease status. Table 1 and Figure 1 show some articles and conclusions regarding transposon mutagenesis applied to oral streptococci.