Method For Transposon Mutagenesis Research Articles

Single-cell genomics analysis reveals complex genetic interactions in an in vivo model of acquired BRAF inhibitor resistance.

The evolution of therapeutic resistance is a major obstacle to the success of targeted oncology drugs. While both inter- and intratumoral heterogeneity limit our ability to detect resistant subpopulations that pre-exist or emerge during treatment, our ability to analyze tumors with single-cell resolution is limited. Here, we utilized a cell-based transposon mutagenesis method to identify mechanisms of BRAF inhibitor resistance in a model of cutaneous melanoma. This screen identified overexpression of NEDD4L and VGLL3 as significant drivers of BRAF inhibitor resistance in vivo. In addition, we describe a novel single-cell genomics profiling method to genotype thousands of individual cells within tumors driven by transposon mutagenesis. This approach revealed a surprising genetic diversity among xenograft tumors and identified recurrent co-occurring mutations that emerge within distinct tumor subclones. Taken together, these observations reveal an unappreciated genetic complexity that drives BRAF inhibitor resistance.

Genetic analysis of electroactive biofilms

Geobacter biofilms synthesize an electroactive exopolysaccharide matrix with conductive pili and c-cytochromes that spatially organizes cells optimally for growth and electron transport to iron oxide substrates, soluble metal contaminants, and current-harvesting electrodes. Despite its relevance to bioremediation and bioenergy applications, little is known about the developmental stages leading to the formation of mature (>20 μm thick) electroactive biofilms. Thus, we developed a transposon mutagenesis method and a high-throughput screening assay and identified mutants of Geobacter sulfurreducens PCA interrupted in the initial stages of surface colonization (attachment and monolayer formation) and the vertical growth and maturation of multilayered biofilms. The molecular dissection of biofilm formation demonstrated that cells undergo a regulated developmental program to first colonize the surface to saturation and then synthesize an electroactive matrix to support optimal cell growth within structured communities. Transitioning from a monolayer to a multilayered, mature biofilm required the expression of conductive pili, consistent with the essential role of these extracellular protein appendages as electronic conduits across all layers of the biofilms. The genetic screening also identified cell envelope processes, regulatory pathways, and electron transport components not previously linked to biofilm formation. These genes provide much-needed understanding of the cellular reprogramming needed to build electroactive biofilms. Importantly, they serve as predictive markers of the physiology and reductive capacity of Geobacter biofilms during the bioremediation of toxic metals and radionuclides and current harvesting in bioelectrochemical systems.

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing.

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 Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

Use of Genetically Modified Bacteria to Repair Cracks in Concrete.

In this paper, we studied the crack-repair by spraying bacteria-based liquid around the cracks in concrete. To enhance the repair efficiency and speed up the repair process, the transposon mutagenesis method was employed to modify the genes of Bacillus halodurans and create a mutant bacterial strain with higher efficiency of calcium carbonate productivity by catalyzing the combination of carbonate and calcium ion. The efficiency of crack-repairing in concrete by spraying two kinds of bacterial liquid was evaluated via image analysis, X-ray computed tomography (X-CT) scanning technology and the sorptivity test. The results show that the crack-repair efficiency was enhanced very evidently by spraying genetically modified bacterial-liquid as no microbiologically induced calcite precipitation (MICP) was found within the cracks for concrete samples sprayed using wild type bacterial-liquid. In addition, the crack-repair process was also shortened significantly in the case of genetically modified bacteria.

A simplified transposon mutagenesis method to perform phenotypic forward genetic screens in cultured cells

BackgroundThe introduction of genome-wide shRNA and CRISPR libraries has facilitated cell-based screens to identify loss-of-function mutations associated with a phenotype of interest. Approaches to perform analogous gain-of-function screens are less common, although some reports have utilized arrayed viral expression libraries or the CRISPR activation system. However, a variety of technical and logistical challenges make these approaches difficult for many labs to execute. In addition, genome-wide shRNA or CRISPR libraries typically contain of hundreds of thousands of individual engineered elements, and the associated complexity creates issues with replication and reproducibility for these methods.ResultsHere we describe a simple, reproducible approach using the SB transposon system to perform phenotypic cell-based genetic screens. This approach employs only three plasmids to perform unbiased, whole-genome transposon mutagenesis. We also describe a ligation-mediated PCR method that can be used in conjunction with the included software tools to map raw sequence data, identify candidate genes associated with phenotypes of interest, and predict the impact of recurrent transposon insertions on candidate gene function. Finally, we demonstrate the high reproducibility of our approach by having three individuals perform independent replicates of a mutagenesis screen to identify drivers of vemurafenib resistance in cultured melanoma cells.ConclusionsCollectively, our work establishes a facile, adaptable method that can be performed by labs of any size to perform robust, genome-wide screens to identify genes that influence phenotypes of interest.

The reaction of soybean symbiotic apparatus to losses of water content in leaves and roots, induced by continuous action of drought

The reaction of the soybean symbiotic apparatus inoculated with Bradyrhizobium japonicum strains and Tn5 mutants, which were different in efficiency, was studied for the loss of water content in leaves and roots induced by prolonged drought, as well as the seed productivity of the formed symbiotic systems. To conduct researches were used microbiological, physiological, biochemical methods, gas chromatography and spectrophotometry. The objects of the study were selected symbiotic systems formed with the participation of soybean plants and strains B. japonicum 646 (active, virulent) and 604k (inactive, highly virulent), as well as Tn5-mutants – B1-20 (active, virulent) and 107 (low-active, virulent) obtained by the method of transposon mutagenesis in the department of symbiotic nitrogen fixation at the Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine. Before sowing, sterilized with 70 % ethanol and washed under running water for 1 h, the seeds were inoculated with suspensions of nodules bacteria (the titre of the suspension was 108 cells in 1 ml). The combined model drought was created during 12 days by stopping watering of plants up to 40 % of full moisture content starting from the stage of two true leaves and gradual transfer of watering to 30 % of full moisture content in the stage of three true leaves and budding - the beginning of flowering. After the stopping of drought, the moisture content of the substrate was adjusted to 60 % of full moisture content (watering recovery) into the stage of mass flowering. Control plants were inoculated by rhizobium culture, as well as plants without inoculation, which grew for optimal watering. It was investigated that in symbiotic systems formed with the participation of soybean and the active strain B. japonicum (646) and Tn5-mutant (B1-20) there was no significant reduction in the water content of plants under drought conditions and the effective work of the symbiotic apparatus was recorded, which contributed to the preservation of seed productivity. Ineffective symbiotic systems observed significant losses in water content and inhibition of the process of nodulation (strain 604k) and nitrogen fixation (Tn5-mutants 107), which was accompanied by significant losses of soybean crop yields. As a result of the research, it was concluded that in soil-climatic conditions with insufficient rainfall and frequent droughts, effective symbiotic systems should be used, which will promote the optimal functioning of the symbiotic apparatus and preserve the seed productivity of soybeans by adaptive regulation of water balance and fixation of molecular nitrogen of the atmosphere. The study of the functioning of leguminous plants in symbiosis with strains of nodule bacteria is important for finding effective symbiotic systems that are able to realize their adaptive potential for the effects of stress factors, in particular drought. Effective symbiotic relationships are the main source of nitrogen fixation in terrestrial ecosystems, which will reduce the need to enrich the soil with chemical compounds and provide additional economic and environmental advantage.

Methods for Transposon Mutagenesis in Proteus mirabilis.

Several methods for transposon mutagenesis have been employed for use in P. mirabilis. The first method involves the use of mini-Tn5 derivatives, which are delivered by conjugation of a suicide plasmid containing this transposon, followed by transposition into the chromosome. A second method is the use of preformed transposon/transposase complexes (transposomes), which are introduced into P. mirabilis cells by electroporation. Each of these methods will be discussed along with the advantages and disadvantages of each.

Transposon Mutagenesis of Bacteroides fragilis.

Bacteroides fragilis is Gram-negative obligatory anaerobe which usually resides in the gut of humans and animals. As an important member of the human gut microbiota it plays a vital role in digestion and absorption of nutrients as well as shaping of host immune system. B. fragilis is also infamous for causing serious infections. Treatment of B. fragilis infections caused emergence of multidrug-resistant strains. Molecular biology tools such as transposon mutagenesis help to decipher and understand commensal and pathogenic faces of B. fragilis. Using two mariner transposon vectors we describe the detailed methodology for the transposon mutagenesis of B. fragilis. We also describe two methods for the identification of transposon integration site (TIS) in transposon mutants. Transposon mutagenesis methods described in this chapter serve as a great tool for studying B. fragilis.

A streamlined method for transposon mutagenesis of Rickettsia parkeri yields numerous mutations that impact infection.

The rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular bacteria, rickettsiae are highly adapted to living inside a variety of host cells, including vascular endothelial cells during mammalian infection. Although it is assumed that the rickettsiae produce numerous virulence factors that usurp or disrupt various host cell pathways, they have been challenging to genetically manipulate to identify the key bacterial factors that contribute to infection. Motivated to overcome this challenge, we sought to expand the repertoire of available rickettsial loss-of-function mutants, using an improved mariner-based transposon mutagenesis scheme. Here, we present the isolation of over 100 transposon mutants in the spotted fever group species Rickettsia parkeri. Transposon insertions disrupted genes whose products are implicated in a variety of pathways, including bacterial replication and metabolism, the type IV secretion system, factors with previously established roles in host cell interactions and pathogenesis, or are of unknown function. Given the need to identify critical virulence factors, forward genetic screens such as this will provide an excellent platform to more directly investigate rickettsial biology and pathogenesis.

Essential Two-Component Systems Regulating Cell Envelope Functions: Opportunities for Novel Antibiotic Therapies.

Novel therapies are urgently needed toalleviate the current crisis of multiple drug-resistant infections.The bacterial signal transduction mechanisms, known as two-component systems (TCSs), are ideal targets of novel inhibitory molecules. Highly restricted to the bacterial world, TCSs control a diverse set of cellular functions, namely virulence, response to cell envelope stress, and drug efflux. Impaired regulation of any of these aspects could affect the susceptibility of bacterial pathogens to antibiotics, which highlights the potential of TCS as targets of antibiotic adjuvant therapies. Moreover, new high-density transposon mutagenesis methods have revealed the existence of TCSs required for growth and viability. Experimental validation of gene essentiality and phenotypic characterization of knockdown mutants indicate that essential TCSs regulate aspects of the cell envelope homeostasis in coordination with cell division. In this review, we describe essential TCSs, and their potentials as targets for antibacterial molecules. We also discuss methods for the identification of small molecules that inhibit TCSs and possible reasons why antibacterial molecules targeting essential TCSs have not yet reached clinical trials.

Poly(A) polymerase I participates in the indole regulatory pathway of Pantoea agglomerans YS19.

Pantoea agglomerans YS19 is a preponderant endophytic bacterium isolated from rice. It is characterized by the formation of symplasmata, a type of multicellular aggregate structure, contributing to a strong stress resistance and specific adaptation of YS19 in endophyte-host associations. Indole is an important signal molecule in intra- or interspecies relationships, regulating a variety of bacterial behaviours such as cell aggregation and stress resistance; however, the regulatory mechanism remains an ongoing area of investigation. This study selected YS19 as a model strain to construct a mutant library, utilizing the mTn5 transposon mutagenesis method, thus obtaining a positive mutant with an indole-inhibited mutation gene. Via thermal asymmetric interlaced PCR, the mutational site was identified as the gene of pcnB, which encodes the poly(A) polymerase I to catalyse the polyadenylation of RNAs. The full length of the pcnB sequence was 1332 bp, and phylogenetic analysis revealed that pcnB is extremely conserved among strains of P. agglomerans. The expression of the gene was significantly inhibited (by 36.6 % as detected via quantitative PCR) by indole (0.5 mM). Many physiological behaviours of YS19 were affected by this mutation: the cell decay rate in the post-stationary growth phase was promoted, symplasmata formation and motility were inhibited in the late stationary growth phase and the colonization ability and growth-promoting effect of YS19 on the host plant were also inhibited. This study discusses the indole regulatory pathways from the point of RNA post-transcriptional modification, thus enriching our knowledge of polyadenylation and expanding current research ideas of indole regulation.

Application of In Vitro Transposon Mutagenesis to Erythromycin Strain Improvement in Saccharopolyspora erythraea.

Transposon mutagenesis is an invaluable technique in molecular biology for the creation of random mutations that can be easily identified and mapped. However, in the field of microbial strain improvement, transposon mutagenesis has scarcely been used; instead, chemical and physical mutagenic methods have been traditionally favored. Transposons have the advantage of creating single mutations in the genome, making phenotype to genotype assignments less challenging than with traditional mutagens which commonly create multiple mutations in the genome. The site of a transposon mutation can also be readily mapped using DNA sequencing primer sites engineered into the transposon termini. In this chapter an in vitro method for transposon mutagenesis of Saccharopolyspora erythraea is presented. Since in vivo transposon tools are not available for most actinomycetes including S. erythraea, an in vitro method was developed. The in vitro method involves a significant investment in time and effort to create the mutants, but once the mutants are made and screened, a large number of highly relevant mutations of direct interest to erythromycin production can be found.

A facile and efficient transposon mutagenesis method for generation of multi-codon deletions in protein sequences

Substitutions, insertions and deletions are all important mutation events in natural and laboratory protein evolution. However, protein engineering using insertions and deletions (indels) is hindered by the lack of a convenient mutagenesis method. Here, we describe a general transposon mutagenesis method that allows for removal of up to five consecutive in-frame codons from a random position of a target protein. This method, referred to as codon deletion mutagenesis (CDM), relies on an engineered Mu transposon that carries asymmetric terminal sequences flanking the MuA transposase recognition sites. CDM requires minimal DNA manipulations, and can generate multi-codon deletions with high efficiency (>90%). As a proof of principle, we constructed five libraries of green fluorescent protein (GFP) containing one to five random codon deletions, respectively. Several variants with multi-codon deletions remained fluorescent, none of which could be easily identified using traditional mutagenesis method. CDM provides a facile and efficient approach to sampling a protein sequence with multi-codon deletions. It will not only facilitate our understanding of the effects of amino acid deletions on protein function but also expedite protein engineering using deletion mutagenesis.

Genome-wide synthetic genetic screening by transposon mutagenesis in Candida albicans.

Transposon-based mutagenesis is an effective method for genetic screening on a genome-wide scale, with particular applicability in organisms possessing compact genomes where transforming DNA tends to integrate by homologous recombination. Methods for transposon mutagenesis have been applied with great success in the budding yeast Saccharomyces cerevisiae and in the related pathogenic yeast Candida albicans. In C. albicans, we have implemented transposon mutagenesis to generate heterozygous mutations for the analysis of complex haploinsufficiency, a type of synthetic genetic interaction wherein a pair of non-complementing heterozygous mutations results in a stronger phenotype then either individual mutation in isolation. Genes exhibiting complex haploinsufficiency typically function within a regulatory pathway, in parallel pathways, or in parallel branches within a single pathway. Here, we present protocols to implement transposon mutagenesis for complex haploinsufficiency screening in C. albicans, indicating methods for transposon construction, mutagenesis, phenotypic screening, and identification of insertion sites in strains of interest. In total, the approach is a useful means to implement large-scale synthetic genetic screening in the diploid C. albicans.

Transposon mutagenesis of the extremely thermophilic bacterium Thermus thermophilus HB27.

Thermus thermophilus is an extremely thermophilic bacterium that grows between 50 and 80°C and is an excellent model organism not only for understanding life at high temperature but also for its biotechnological and industrial applications. Multiple molecular capabilities are available including targeted gene inactivation and the use of shuttle plasmids that replicate in T. thermophilus and Escherichia coli; however, the ability to disrupt gene function randomly by transposon insertion has not been developed. Here we report a detailed method of transposon mutagenesis of T. thermophilus HB27 based on the EZ-Tn5 system from Epicentre Biotechnologies. We were able to generate insertion mutations throughout the chromosome by in vitro transposition and transformation with mutagenized genomic DNA. We also report that an additional step, one that fills in single stranded gaps in donor DNA generated by the transposition reaction, was essential for successful mutagenesis. We anticipate that our method of transposon mutagenesis will enable further genetic development of T. thermophilus and may also be valuable for similar endeavors with other under-developed organisms.

Transposon mutagenesis of the anaerobic commensal, Bacteroides fragilis, using the EZ::TN5 transposome

Genetic analysis of Bacteroides fragilis (BF) is hindered because of the lack of efficient transposon mutagenesis methods. Here, we describe a simple method for transposon mutagenesis using EZ::TN5, a commercially available system that we optimized for use in BF638R. The modified EZ::TN5 transposon contains an Escherichia coli conditional origin of replication, a kanamycin resistance gene for E. coli, an erythromycin resistance gene for BF , and 19 basepair transposase recognition sequences on either ends. Electroporation of the transposome (transposon-transposase complex) into BF638R yielded 3.2 ± 0.35 × 10(3) CFU μg(-1) of transposon DNA. Modification of the transposon by the BF638R restriction/modification system increased transposition efficiency sixfold. Electroporation of the EZ::TN5 transposome results in a single-copy insertion of the transposon evenly distributed across the genome of BF638R and can be used to construct a BF638R transposon library. The transposon was also effective in mutating a BF clinical isolate and a strain of the related species, Bacteroides thetaiotaomicron. The EZ::TN5-based mutagenesis described here is more efficient than other transposon mutagenesis approaches previously reported for BF.

In vivo and in silico determination of essential genes of Campylobacter jejuni

BackgroundIn the United Kingdom, the thermophilic Campylobacter species C. jejuni and C. coli are the most frequent causes of food-borne gastroenteritis in humans. While campylobacteriosis is usually a relatively mild infection, it has a significant public health and economic impact, and possible complications include reactive arthritis and the autoimmune diseases Guillain-Barré syndrome. The rapid developments in "omics" technologies have resulted in the availability of diverse datasets allowing predictions of metabolism and physiology of pathogenic micro-organisms. When combined, these datasets may allow for the identification of potential weaknesses that can be used for development of new antimicrobials to reduce or eliminate C. jejuni and C. coli from the food chain.ResultsA metabolic model of C. jejuni was constructed using the annotation of the NCTC 11168 genome sequence, a published model of the related bacterium Helicobacter pylori, and extensive literature mining. Using this model, we have used in silico Flux Balance Analysis (FBA) to determine key metabolic routes that are essential for generating energy and biomass, thus creating a list of genes potentially essential for growth under laboratory conditions. To complement this in silico approach, candidate essential genes have been determined using a whole genome transposon mutagenesis method. FBA and transposon mutagenesis (both this study and a published study) predict a similar number of essential genes (around 200). The analysis of the intersection between the three approaches highlights the shikimate pathway where genes are predicted to be essential by one or more method, and tend to be network hubs, based on a previously published Campylobacter protein-protein interaction network, and could therefore be targets for novel antimicrobial therapy.ConclusionsWe have constructed the first curated metabolic model for the food-borne pathogen Campylobacter jejuni and have presented the resulting metabolic insights. We have shown that the combination of in silico and in vivo approaches could point to non-redundant, indispensable genes associated with the well characterised shikimate pathway, and also genes of unknown function specific to C. jejuni, which are all potential novel Campylobacter intervention targets.

Effective mutagenesis of Vibrio fischeri by using hyperactive mini-Tn5 derivatives.

We have developed a transposon mutagenesis system for Vibrio fischeri ES114 that utilizes a hyperactive mutant Tn5 transposase (E54K and M56A) and optimized transposon ends. Using a conjugation-based procedure, we obtained independent single-insertion mini-Tn5 mutants at a rate of approximately 10(-6). This simple and inexpensive technique represents a significant improvement over previous methods for transposon mutagenesis of V. fischeri and should be applicable to many other bacteria.

Construction of a Large Signature-Tagged Mini-Tn 5 Transposon Library and Its Application to Mutagenesis of Sinorhizobium meliloti

Sinorhizobium meliloti genome sequence determination has provided the basis for different approaches of functional genomics for this symbiotic nitrogen-fixing alpha-proteobacterium. One of these approaches is gene disruption with subsequent analysis of mutant phenotypes. This method is efficient for single genes; however, it is laborious and time-consuming if it is used on a large scale. Here, we used a signature-tagged transposon mutagenesis method that allowed analysis of the survival and competitiveness of many mutants in a single experiment. A novel set of signature tags characterized by similar melting temperatures and G+C contents of the tag sequences was developed. The efficiencies of amplification of all tags were expected to be similar. Thus, no preselection of the tags was necessary to create a library of 412 signature-tagged transposons. To achieve high specificity of tag detection, each transposon was bar coded by two signature tags. In order to generate defined, nonredundant sets of signature-tagged S. meliloti mutants for subsequent experiments, 12,000 mutants were constructed, and insertion sites for more than 5,000 mutants were determined. One set consisting of 378 mutants was used in a validation experiment to identify mutants showing altered growth patterns.

Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning.

We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening approximately 38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.