Mutagenesis System Research Articles

Engineered Zymomonas mobilis for salt tolerance using EZ-Tn5-based transposon insertion mutagenesis system.

BackgroundThe cell growth and ethanol yield of Zymomonas mobilis may be detrimentally affected by salt stress frequently present in some biomass-based fermentation systems, leading to a decrease in the rate of sugar conversion to ethanol or other bioproducts. To address this problem, improving the salt tolerance of Z. mobilis is a desirable way. However, limited progress has been made in development of Z. mobilis with higher salt tolerance for some technical challenges in the past decades. Recently, transposon insertion mutant system has been widely used as a novel genetic tool in many organisms to develop mutant strains. In this study, Tn5-based transposon insertion mutagenesis system firstly used for construction of higher salt tolerance strain in Z. mobilis.ResultsApproximately 200 Z. mobilis ZM4 mutants were generated by using Tn5-based transposon mutagenesis system. The mutant strain ZMT2 with improved salt tolerance phenotype was obtained by screening on RM agar plates with additional 1 % NaCl. Strain ZMT2 was confirmed to exhibit better fermentation performance under NaCl stress than wild type of strain ZM4. The transposon insertion was located in ZMO1122 (himA) by genome walking. Discruption of himA gene showed that himA may play an important role in response to salt tolerance in Z. mobils.ConclusionsThe mutant strain ZMT2 with a transposon insertion in himA gene of the genome showed obviously higher sugar conversion rate to ethonal under up to 2 % NaCl stress than did the wild ZM4 strain. Besides, ZMT2 exhibited shared fermentative capabilities with wild ZM4 strain under no or low NaCl stress. This report firstly showed that himA played a role in responding to NaCl stress. Furthermore, the result indicated that Tn5-based transposon mutagenesis system was a feasible tool not only for genetic engineering in Z. mobilis strain improvement, but also in tapping resistent genes.

An influence of the copy number of biosynthetic gene clusters on the production level of antibiotics in a heterologous host

Streptomyces albus J1074 is a well-known host for heterologous expression of secondary metabolites. To further increase its potential and to study the influence of cluster multiplication, additional φC31-attachment site was integrated into its genome using a system for transposon mutagenesis. Four secondary metabolite clusters were expressed in strains with different numbers of attachment sites, ranging from one to three copies of the site. Secondary metabolite production was examined and a new compound could be detected, purified and its structure was elucidated.

Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis.

Next generation sequencing (NGS) technologies have dramatically improved studies in biology and biomedical science. However, no optimal NGS approach is available to conveniently analyze low frequency mutations caused by DNA damage treatments. Here, by developing an exquisite ultra-sensitive NGS (USNGS) platform “EasyMF” and incorporating it with a widely used supF shuttle vector-based mutagenesis system, we can conveniently dissect roles of lesion bypass polymerases in damage-induced mutagenesis. In this improved mutagenesis analysis pipeline, the initial steps are the same as in the supF mutation assay, involving damaging the pSP189 plasmid followed by its transfection into human 293T cells to allow replication to occur. Then “EasyMF” is employed to replace downstream MBM7070 bacterial transformation and other steps for analyzing damage-induced mutation frequencies and spectra. This pipeline was validated by using UV damaged plasmid after its replication in lesion bypass polymerase-deficient 293T cells. The increased throughput and reduced cost of this system will allow us to conveniently screen regulators of translesion DNA synthesis pathway and monitor environmental genotoxic substances, which can ultimately provide insight into the mechanisms of genome stability and mutagenesis.

Single-Plasmid-Based System for Efficient Noncanonical Amino Acid Mutagenesis in Cultured Mammalian Cells.

We describe a new expression system for efficient non-canonical amino acid mutagenesis in cultured mammalian cells by using the pyrrolysine tRNA synthetase/tRNACUA (Pyl) pair. A significant improvement in the incorporation of non-canonical amino acids into proteins was obtained by combining all the required genetic components into a single and compact vector that can be efficiently delivered to different mammalian cell lines by conventional transfection reagents.

An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.

Multiplex conditional mutagenesis in zebrafish using the CRISPR/Cas system.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a powerful tool for genome editing in numerous organisms. However, the system is typically used for gene editing throughout the entire organism. Tissue and temporal specific mutagenesis is often desirable to determine gene function in a specific stage or tissue and to bypass undesired consequences of global mutations. We have developed the CRISPR/Cas system for conditional mutagenesis in transgenic zebrafish using tissue-specific and/or inducible expression of Cas9 and U6-driven expression of sgRNA. To allow mutagenesis of multiple targets, we have isolated four distinct U6 promoters and designed Golden Gate vectors to easily assemble transgenes with multiple sgRNAs. We provide experimental details on the reagents and applications for multiplex conditional mutagenesis in zebrafish.

Performance of optimized noncanonical amino acid mutagenesis systems in the absence of release factor 1.

Site-specific incorporation of noncanonical amino acids (ncAAs) into proteins expressed in E. coli using UAG-suppression competes with termination mediated by release factor 1 (RF1). Recently, unconditional deletion of RF1 was achieved in a genomically recoded E. coli (C321), devoid of all endogenous UAG stop codons. Here we evaluate the efficiency of ncAA incorporation in this strain using optimized suppression vectors. Even though the absence of RF1 does not benefit the suppression efficiency of a single UAG codon, multi-site incorporation of a series of chemically distinct ncAAs was significantly improved.

A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria.

Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.

Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus

Filamentous fungi have a dominant nonhomologous-end joining (NHEJ) DNA repair pathway, which results in the majority of transformed progenies having random heterologous insertion mutagenesis. Thus, lack of a versatile genome-editing tool prevents us from carrying out precise genome editing to explore the mechanism of pathogenesis. Moreover, clinical isolates that have a wild-type ku80 background without any selection nutrition marker especially suffer from low homologous integration efficiency. In this study, we have established a highly efficient CRISPR mutagenesis system to carry out precise and efficient in-frame integration with or without marker insertion with approximately 95–100% accuracy via very short (approximately 35-bp) homology arms in a process referred to as microhomology-mediated end joining (MMEJ). Based on this system, we have successfully achieved an efficient and precise integration of an exogenous GFP tag at the predicted site without marker insertion and edited a conidial melanin gene pksP and a catalytic subunit of calcineurin gene cnaA at multiple predicted sites with or without selection marker insertion. Moreover, we found that MMEJ-mediated CRISPR-Cas9 mutagenesis is independent of the ku80 pathway, indicating that this system can function as a powerful and versatile genome-editing tool in clinical Aspergillus isolates.

NHL and RCGA Based Multi-Relational Fuzzy Cognitive Map Modeling for Complex Systems

In order to model multi-dimensions and multi-granularities oriented complex systems, this paper firstly proposes a kind of multi-relational Fuzzy Cognitive Map (FCM) to simulate the multi-relational system and its auto construct algorithm integrating Nonlinear Hebbian Learning (NHL) and Real Code Genetic Algorithm (RCGA). The multi-relational FCM fits to model the complex system with multi-dimensions and multi-granularities. The auto construct algorithm can learn the multi-relational FCM from multi-relational data resources to eliminate human intervention. The Multi-Relational Data Mining (MRDM) algorithm integrates multi-instance oriented NHL and RCGA of FCM. NHL is extended to mine the causal relationships between coarse-granularity concept and its fined-granularity concepts driven by multi-instances in the multi-relational system. RCGA is used to establish high-quality high-level FCM driven by data. The multi-relational FCM and the integrating algorithm have been applied in complex system of Mutagenesis. The experiment demonstrates not only that they get better classification accuracy, but it also shows the causal relationships among the concepts of the system.

Development of potent in vivo mutagenesis plasmids with broad mutational spectra.

Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in <24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system also enables the continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in <10 h. Our findings enable broad-spectrum mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms.

Sleeping Beauty Transposon Mutagenesis as a Tool for Gene Discovery in the NOD Mouse Model of Type 1 Diabetes.

A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified >40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying “natural” alleles in the human population is to engineer “artificial” alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis.

An in vitro recombination-based reverse genetic system for rapid mutagenesis of structural genes of the Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is one of the most common pathogens of severe viral encephalitis, which is a severe threat to human health. Despite instability of the JEV genome in bacteria, many strategies have been developed to establish molecular clone systems of JEV, providing convenient tools for studying the virus life cycle and virus-host interactions. In this study, we adapted an In-Fusion enzyme-based in vitro recombination method to construct a reverse genetic system of JEV, thereby providing a rapid approach to introduce mutations into the structural genes. A truncated genome without the structural genes was constructed as the backbone, and the complementary segment containing the structural genes was recombined in vitro, which was then transfected directly into virus-permissive cells. The progeny of the infectious virus was successfully detected in the supernatant of the transfected cells, and showed an identical phenotype to its parental virus. To provide a proof-of-principle, the 12 conserved cysteine residues in the envelope (E) protein of JEV were respectively mutated using this approach, and all mutations resulted in a complete failure to generate infectious virus. However, a leucine-tophenylanine mutation at amino acid 107 of the E protein did not interfere with the production of the infectious virus. These results suggested that all 12 cysteines in the E protein are essential for the JEV life cycle. In summary, a novel reverse genetic system of JEV was established for rapidly introducing mutations into structural genes, which will serve as a useful tool for functional studies.

CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening

Site-directed mutagenesis using genetic approaches can provide a wealth of resources for crop breeding as well as for biological research. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 endonuclease (CRISPR/Cas9) system is a novel strategy used to induce mutations in a specific genome region; the system functions in a variety of organisms, including plants. Here, we report application of the CRISPR/Cas9 system to efficient mutagenesis of the tomato genome. In this study, we targeted the tomato RIN gene, which encodes a MADS-box transcription factor regulating fruit ripening. Three regions within the gene were targeted and mutations consisting either of a single base insertion or deletion of more than three bases were found at the Cas9 cleavage sites in T0 regenerated plants. The RIN-protein-defective mutants produced incomplete-ripening fruits in which red color pigmentation was significantly lower than that of wild type, while heterologous mutants expressing the remaining wild-type gene reached full-ripening red color, confirming the important role of RIN in ripening. Several mutations that were generated at three independent target sites were inherited in the T1 progeny, confirming the applicability of this mutagenesis system in tomato.

Acinetobacter baumannii Virulence Is Mediated by the Concerted Action of Three Phospholipases D.

Acinetobacter baumannii causes a broad range of opportunistic infections in humans. Its success as an emerging pathogen is due to a combination of increasing antibiotic resistance, environmental persistence and adaptation to the human host. To date very little is known about the molecular basis of the latter. Here we demonstrate that A. baumannii can use phosphatidylcholine, an integral part of human cell membranes, as sole carbon and energy source. We report on the identification of three phospholipases belonging to the PLD superfamily. PLD1 and PLD2 appear restricted to the bacteria and display the general features of bacterial phospholipases D. They possess two PLDc_2 PFAM domains each encompassing the HxKx4Dx6GS/GGxN (HKD) motif necessary for forming the catalytic core. The third candidate, PLD3, is found in bacteria as well as in eukaryotes and harbours only one PLDc_2 PFAM domain and one conserved HKD motif, which however do not overlap. Employing a markerless mutagenesis system for A. baumannii ATCC 19606T, we generated a full set of PLD knock-out mutants. Galleria mellonella infection studies as well as invasion experiments using A549 human lung epithelial cells revealed that the three PLDs act in a concerted manner as virulence factors and are playing an important role in host cell invasion.

Mitochondrial Disease-related Mutation G167P in Cytochrome b of Rhodobacter capsulatus Cytochrome bc1 (S151P in Human) Affects the Equilibrium Distribution of [2Fe-2S] Cluster and Generation of Superoxide

Cytochrome bc1 is one of the key enzymes of many bioenergetic systems. Its operation involves a large scale movement of a head domain of iron-sulfur protein (ISP-HD), which functionally connects the catalytic quinol oxidation Qo site in cytochrome b with cytochrome c1. The Qo site under certain conditions can generate reactive oxygen species in the reaction scheme depending on the actual position of ISP-HD in respect to the Qo site. Here, using a bacterial system, we show that mutation G167P in cytochrome b shifts the equilibrium distribution of ISP-HD toward positions remote from the Qo site. This renders cytochrome bc1 non-functional in vivo. This effect is remediated by addition of alanine insertions (1Ala and 2Ala) in the neck region of the ISP subunit. These insertions, which on their own shift the equilibrium distribution of ISP-HD in the opposite direction (i.e. toward the Qo site), also act in this manner in the presence of G167P. Changes in the equilibrium distribution of ISP-HD in G167P lead to an increased propensity of cytochrome bc1 to generate superoxide, which becomes evident when the concentration of quinone increases. This result corroborates the recently proposed model in which "semireverse" electron transfer back to the Qo site, occurring when ISP-HD is remote from the site, favors reactive oxygen species production. G167P suggests possible molecular effects of S151P (corresponding in sequence to G167P) identified as a mitochondrial disease-related mutation in human cytochrome b. These effects may be valid for other human mutations that change the equilibrium distribution of ISP-HD in a manner similar to G167P.

Abstract 4186: p120 catenin: A novel regulator of epithelial cell delamination in early Kras-driven pancreatic cancer

Abstract By 2020, pancreatic cancer is estimated to climb from the 4th to the 2nd most common cause of cancer-related deaths in the United States. This deadly disease has continued to remain largely refractory to chemotherapeutic and treatment regimens, and patients often experience a heavy metastatic burden. A study combining the Sleeping beauty transposon random insertion mutagenesis system with an oncogenic KrasG12D allele in mice as a screen to identify candidate pancreatic cancer genes identified genes enriched in adherens and tight junctions as significantly enriched in Kras-driven neoplasia with Ctnnd1 as a locus commonly mutated in mice developing metastatic progression of pancreatic cancer. Ctnnd1 encodes the adherens junction protein p120 catenin, which is integral in stabilization of cadherin molecules at cell membranes. p120 catenin is misexpressed in an estimated 60% primary pancreatic tumors and low/absent expression levels and predominant cytoplasmic localization of p120 catenin in primary resected pancreatic tumors correlates with worse survival in pancreatic cancer patients. Yet, the mechanisms by which p120 catenin contributes to the pathogenesis of pancreatic cancer are not clear. We have comprehensively examined p120 catenin staining in human Pancreatic Intraepithelial Neoplasia (PanIN) and identified mislocalization of p120 catenin to the cytoplasm as early as PanIN2. 3/5 PanIN3 examined had predominant cytoplasmic staining, which led us to hypothesize that p120 catenin might play a critical role in early pancreatic neoplasia, before the onset of Pancreatic Ductal Adenocarcinoma. To examine the role of p120 catenin during early Kras-driven pancreatic neoplasia, we ablated p120 catenin in a mouse model of preinvasive pancreatic cancer, KCiMist1. KCiMist1p120f/f pancreases display significant acceleration of acinar to ductal metaplasia (ADM) and PanIN formation when compared to KCiMist1p120wt/wt pancreases one month post tamoxifen injection. KCiMist1p120f/f pancreases are significantly larger than KCiMist1p120wt/wt pancreases with 92.89% pancreatic area occupied by Fibrostroma one month post tamoxifen injection. As a result, KCiMist1p120f/f animals have severe exocrine pancreatic insufficiency and die on average 8 weeks earlier than their KCiMist1p120wt/wt controls. Lineage tracing revealed a prominent epithelial cell delamination phenotype in KCiMist1p120f/f pancreases. Quantification of epithelial cells in the extensive stroma revealed a striking 832/7000 CK19+ cells in KCiMist1p120f/f pancreases vs 15/7000 CK19+ cells in KCiMist1p120wt/wt pancreases. Microarray analysis showed &amp;gt;1263 differentially expressed genes! IPA pathway analysis revealed significant gene expression changes in PI3K/AKT and Cdc42 signaling. Taken together, our results suggest a critical role for p120 catenin in regulating epithelial cell delamination in early pancreatic cancer. Citation Format: Audrey M. Hendley, Yue J. Wang, Janivette Alsina, Ishrat Ahmed, Hao Zhang, Samuel Savidge, Hao Ho, Albert Reynolds, Anirban Maitra, Michael Goggins, Christine Iacobuzio-Donahue, Steven D. Leach, Jennifer M. Bailey. p120 catenin: A novel regulator of epithelial cell delamination in early Kras-driven pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4186. doi:10.1158/1538-7445.AM2015-4186

The sactibiotic subclass of bacteriocins: an update.

The sactibiotics are a recently designated subclass of bacteriocins that contain characteristic cysteine sulphur to α -carbon linkages mediated through post-translational modifications. They are a relatively small subclass of bacteriocins compared to the most thoroughly studied lantibiotics. The sactibiotics that have been extensively studied thus far are thuricin CD, subtilosin A, thurincin H, and propionicin F. Despite their recent discovery, there have already been significant advances made in the study of sactibiotics, most notably the discovery of the narrow spectrum anti-Clostridium difficile sactibiotic, thuricin CD. In addition, scientists have gained insights into the mechanisms of action of the sactibiotic subtilosin A, which targets Listeria monocytogenes,Gardnerella vaginalis, and other pathogens. Also, the development of heterologous host systems and homologous expression and site-directed mutagenesis systems for the sactibiotic thurincin H have opened up many opportunities for further studies on this sactibiotic. These and other recent studies concerning the molecular biology, 3D structural elucidation, mode of action, self-protection mechanisms, and antimicrobial spectrum of the sactibiotic subgroup of bacteriocins are discussed in this review.

Conditional U1 Gene Silencing in Toxoplasma gondii.

The functional characterisation of essential genes in apicomplexan parasites, such as Toxoplasma gondii or Plasmodium falciparum, relies on conditional mutagenesis systems. Here we present a novel strategy based on U1 snRNP-mediated gene silencing. U1 snRNP is critical in pre-mRNA splicing by defining the exon-intron boundaries. When a U1 recognition site is placed into the 3’-terminal exon or adjacent to the termination codon, pre-mRNA is cleaved at the 3’-end and degraded, leading to an efficient knockdown of the gene of interest (GOI). Here we describe a simple method that combines endogenous tagging with DiCre-mediated positioning of U1 recognition sites adjacent to the termination codon of the GOI which leads to a conditional knockdown of the GOI upon rapamycin-induction. Specific knockdown mutants of the reporter gene GFP and several endogenous genes of T. gondii including the clathrin heavy chain gene 1 (chc1), the vacuolar protein sorting gene 26 (vps26), and the dynamin-related protein C gene (drpC) were silenced using this approach and demonstrate the potential of this technology. We also discuss advantages and disadvantages of this method in comparison to other technologies in more detail.

A Multifunctional Mutagenesis System for Analysis of Gene Function in Zebrafish.

Since the sequencing of the human reference genome, many human disease-related genes have been discovered. However, understanding the functions of all the genes in the genome remains a challenge. The biological activities of these genes are usually investigated in model organisms such as mice and zebrafish. Large-scale mutagenesis screens to generate disruptive mutations are useful for identifying and understanding the activities of genes. Here, we report a multifunctional mutagenesis system in zebrafish using the maize Ds transposon. Integration of the Ds transposable element containing an mCherry reporter for protein trap events and an EGFP reporter for enhancer trap events produced a collection of transgenic lines marking distinct cell and tissue types, and mutagenized genes in the zebrafish genome by trapping and prematurely terminating endogenous protein coding sequences. We obtained 642 zebrafish lines with dynamic reporter gene expression. The characterized fish lines with specific expression patterns will be made available through the European Zebrafish Resource Center (EZRC), and a database of reporter expression is available online (http://fishtrap.warwick.ac.uk/). Our approach complements other efforts using zebrafish to facilitate functional genomic studies in this model of human development and disease.