Construction of a Large Signature-Tagged Mini-Tn5 Transposon Library and Its Application to Mutagenesis of Sinorhizobium meliloti (original) (raw)
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DNA Research, 2008
Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29 330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G1C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth-and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward-and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology.
Current Opinion in Microbiology, 2005
Signature-tagged mutagenesis (STM) is a powerful negative selection method, predominantly used to identify the genes of a pathogen that are required for the successful colonization of an animal host. Since its first description a decade ago, STM has been applied to screen a vast amount of transposon insertion mutants in 31 bacterial species. This has led to the identification of over 1700 bacterial genes that are involved in virulence. Despite the preservation of the basic design, the STM method has been developed further owing to recent advances including different designs of the signature-tags and profound changes in the mode of detection. These advances promoted substantially the application range and versatility of the STM method.
Systematic Mutagenesis of the Escherichia coli Genome
Journal of Bacteriology, 2004
A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the Red proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition.
Microbiology, 1998
Soil bacteria, such as Sinorhizobium meliloti, are subject to variation in environmental conditions, including carbon-and nitrogen-deprivation. The ability of bacteria to sense changes in their environment and respond accordingly is of vital importance to their survival and persistence in the soil and rhizosphere. A derivative of Tn5 which creates transcriptional fusions to the promoterless luxAB genes was used to mutagenize 5. meliloti 1021 and 5000 insertion mutants were subsequently screened for gene fusions induced by selected environmental stresses. The isolation of 21 gene fusions induced by nitrogen-deprivation and 12 induced by carbon-deprivation is described. Cloning and partial DNA sequence analysis of the transposon-tagged loci revealed a variety of novel genes, as well as S. meliloti genes with significant similarity to known bacterial loci. In addition, nodule occupancy studies were carried out with selected TnSluxAB insertion mutants to examine the role of the tagged genes in competition.
Research in …, 2004
Analysis of the complete DNA sequences of many microbial genomes available reveals a fair number of putative ORFs without an identified function. A systematic scan of the Escherichia coli chromosome was achieved by random transposition with a newly developed Tn5 minitransposon derivative carrying the arabinose-inducible araP BAD promoter oriented outward at one end (Tn5-araP BAD ). The transposon insertion mutants obtained were assayed for conditional lethal phenotypes (arabinose dependence or sensitivity), for growth at two temperatures (37 and 15 • C) and in different media (rich and minimal medium). The Tn5-araP BAD -tagged genes were identified by sequencing the transposon insertion points. In this way we found a new essential gene cluster (yhbN-yhbG), produced conditional lethal (arabinose-dependent) mutations in already known essential genes (folD, frr, plsC, thiL, serS, thrS, and trpS) and provided a new phenotype (cold sensitivity) to other known genes (holD, ahpC, and tolA). Moreover, we identified eight putative ORFs (kch, ycaM, ycbQ, yddA, yddB, ydeK, ydeX, and yliF) that appear to be required in optimum growth conditions (rich medium at 37 • C) but not in the cold and in minimal medium. 2004 Elsevier SAS. All rights reserved.
Applied and Environmental Microbiology, 2006
A simple and high-throughput transposon-mediated mutagenesis system employing two different types of transposons in combination with direct genomic DNA amplification and thermal asymmetric interlaced PCR (TAIL-PCR) was developed. Each of the two minitransposons based on IS31831 (ISL3 family) and Tn5 (IS4 family) was integrated into the Corynebacterium glutamicum R genome. By using BLAST and Perl, transposon insertion locations were automatically identified based on the sequences of TAIL-PCR products of mutant cells. Insertion locations of 18,000 mutants were analyzed, and a comprehensive insertion library covering nearly 80% of the 2,990 open reading frames of C. glutamicum R was generated. Eight thousand of the mutants, exhibiting disruption in 2,330 genes, survived on complex medium under normal laboratory conditions, indicating that the genes were not essential for cell survival. Of the 2,330 genes, 30 exhibited high similarity to essential genes of Escherichia coli or Bacillus subtilis. This approach could be useful in furthering genetic understanding of cellular life and facilitating the functional analysis of microorganisms.
Mini-transposons in microbial ecology and environmental biotechnology
Fems Microbiology Ecology, 1998
Mini-transposon is the generic name given to the members of a collection of genetic assets derived from transposons Tn10 and Tn5, in which the naturally occurring functional segments of DNA have been rearranged artificially to originate shorter mobile elements. In the most widespread design (that known as the pUT system), any heterologous DNA segment can be conveniently cloned within the boundaries of a mini-Tn5 vector and finally inserted into the chromosome of target Gramnegative bacteria after a few simple genetic manipulations. The large variety of antibiotic, non-antibiotic and excisable selection markers available has been combined at ease with DNA fragments encoding one or more phenotypes of interest for ecological or biotechnological applications. These include the tagging of specific strains in a community with selectable and/or optical marker genes, the production of stable gene fusions for monitoring transcriptional regulation in single cells, the metabolic engineering of strains destined for bioremediation, the non-disruptive monitoring of gene transfer and the assembly of gene containment and strain containment circuits for genetically manipulated microorganisms. z
Archives of Microbiology, 2008
Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium, which was originally isolated from the interior of sugarcane plants. The genome of strain PAL5 of G. diazotrophicus has been completely sequenced and a next step is the functional characterization of its genes. The aim of this study was to establish an efficient mutagenesis method, using the commercial Tn5 transposon EZ::Tn5™Tnp Transposome™ (Epicentre). Up to 1 × 106 mutants per microgram of transposome were generated in a single electroporation experiment. Insertion-site flanking sequences were amplified by inverse PCR and sequenced for 31 mutants. For ten of these mutants, both insertion flanks could be identified, confirming the 9 bp duplication that is typical for Tn5 transposition. Insertions occurred in a random fashion and were genetically stable for at least 50 generations. One mutant had an insertion in a homolog of the flagellar gene flgA, and was therefore predicted to be affected in flagella-dependent traits and used to validate the applied mutagenesis methodology. This mutant lacked flagella and was non-motile on soft agar. Interestingly, it was also strongly affected in the ability to form biofilm on glass wool.
Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing
Journal of Visualized Experiments, 2020
Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The "last-line" antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gramnegative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.
Applications of Transposon-Based Gene Delivery System in Bacteria
Journal of Microbiology and Biotechnology, 2009
Mobile genetic segments, or transposons, are also referred to as "jumping genes" as they can shift from one position in the genome to another, thus inducing a chromosomal mutation. According to the target site-specificity of the transposon during a transposition event, the result is either the insertion of a gene of interest at a specific chromosomal site, or the creation of knockout mutants. The former situation includes the integration of conjugative transposons via site-specific recombination, several transposons preferring a target site of a conserved AT-rich sequence, and Tn7 being site-specifically inserted at attTn7, the downstream of the essential glmS gene. The latter situation is exploited for random mutagenesis in many prokaryotes, including IS (insertion sequence) elements, mariner, Mu, Tn3 derivatives (Tn4430 and Tn917), Tn5, modified Tn7, Tn10, Tn552, and Ty1, enabling a variety of genetic manipulations. Randomly inserted transposons have been previously employed for a variety of applications such as genetic footprinting, gene transcriptional and translational fusion, signaturetagged mutagenesis (STM), DNA or cDNA sequencing, transposon site hybridization (TraSH), and scanning linker mutagenesis (SLM). Therefore, transposon-mediated genetic engineering is a valuable discipline for the study of bacterial physiology and pathogenesis in living hosts.