ABSTRACT: This project tested if siderophores are publig goods in Pseudomonas, and involved resequencing a well used siderophore mutant made by non-specific mutagenesis techniques
Project description:Pseudomonas species are ubiquitous in plant-associated environments and produce an array of volatiles, enzymes and antimicrobials. The biosynthesis of many metabolites is regulated by the GacS/GacA two-component regulatory system. Transcriptome analysis of Pseudomonas fluorescens SBW25 revealed that 702 genes were differentially regulated (fold change>4, P<0.0001) in a gacS::Tn5 mutant, with 300 and 402 genes up- and down-regulated, respectively. Genes that were significantly down-regulated are involved in viscosin biosynthesis (viscABC), protease production (aprA), motility, biofilm formation, and secretory systems. Genes that were significantly up-regulated are involved in siderophore biosynthesis and oxidative stress. In contrast to previous studies with gac-mutants of other Pseudomonas species/strains, the gacS mutant of SBW25 inhibited growth of oomycete, fungal and bacterial pathogens significantly more than parental strain SBW25. A potential candidate for this enhanced antimicrobial activity was a large nonribosomal peptide synthetase (NRPS) gene cluster predicted to encode for an 8-amino-acid ornicorrugatin-like peptide. Site-directed mutagenesis of an NRPS gene in this cluster, however, did not lead to a reduction in the antimicrobial activity of the gacS mutant. Collectively these results indicate that a mutation in the GacS/GacA regulatory system causes major transcriptional changes in P. fluorescens SBW25 and significantly enhances its antimicrobial activities by yet unknown mechanisms. This expression study used total RNA recovered from four separate wild-type cultures of Pseudomonas fluorescens SBW25 and four separate cultures of the gacS mutant. Expression design was based on the updated genome sequence of Pseudomonas fluorescens SBW25, NC_012660.1 and associated plasmid pQBR0476 with nineteen 60-mer probe per gene. Each probe is replicated 3 times. The design includes random GC and other control probes.
Project description:We used three M. tuberculosis strains (WT H37Rv, an RV2752c deletion mutation, and the mutant complemented with an Rv2752c overexpression construct), extracted RNA from log phase cultures, and made RNAseq expression libraries as well as 5'-end-directed libraries.
Project description:Pseudomonas species are ubiquitous in plant-associated environments and produce an array of volatiles, enzymes and antimicrobials. The biosynthesis of many metabolites is regulated by the GacS/GacA two-component regulatory system. Transcriptome analysis of Pseudomonas fluorescens SBW25 revealed that 702 genes were differentially regulated (fold change>4, P<0.0001) in a gacS::Tn5 mutant, with 300 and 402 genes up- and down-regulated, respectively. Genes that were significantly down-regulated are involved in viscosin biosynthesis (viscABC), protease production (aprA), motility, biofilm formation, and secretory systems. Genes that were significantly up-regulated are involved in siderophore biosynthesis and oxidative stress. In contrast to previous studies with gac-mutants of other Pseudomonas species/strains, the gacS mutant of SBW25 inhibited growth of oomycete, fungal and bacterial pathogens significantly more than parental strain SBW25. A potential candidate for this enhanced antimicrobial activity was a large nonribosomal peptide synthetase (NRPS) gene cluster predicted to encode for an 8-amino-acid ornicorrugatin-like peptide. Site-directed mutagenesis of an NRPS gene in this cluster, however, did not lead to a reduction in the antimicrobial activity of the gacS mutant. Collectively these results indicate that a mutation in the GacS/GacA regulatory system causes major transcriptional changes in P. fluorescens SBW25 and significantly enhances its antimicrobial activities by yet unknown mechanisms.
Project description:The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.
Project description:Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest. Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest.
Project description:We identified a 14-3-3 homolog (maf1) in an EST library made under conditions conducive to aflatoxin biosynthesis. Disruption of this gene in A. flavus by site directed mutagenesis abolished aflatoxin production. The maf1 mutant was morphologically similar to wild type but had reduced conidiation and growth on some media. DNA expression analysis of the wild type and maf1 mutant revealed that the mutation affected the expression profile of a set of genes associated with aflatoxin production. Keywords = Aspergillus Keywords = aflatoxin Keywords = 14-3-3 Keywords: repeat sample
Project description:The aim of the experiment was to identify genes differentially expressed between the susceptible wild type strain P. aeruginosa PAO1 (PT5) and a mutant resistant to a drug-siderophore conjugate, in order to obtain information on the resistance mechanism(s). A mutant of PT5 able to grow at 4 mg/l BAL30072, a drug-siderophore conjugate, was selected in vitro . The susceptible wild type strain PT5 and the mutant (BAL6) were grown in LB medium and the mutant also in the presence of 4 mg/l BAL30072 to mid-exponential growth phase (OD600 =2) in triplicate cultures. RNA was extracted using the RNeasy Kit (Qiagen). A total of nine Affymterix P. aeruginosa arrays were hybridized (one for each replicate) under standard conditions.
Project description:Phenotype-driven forward genetic experiments are among the most powerful approaches for linking biology and disease to genomic elements. Although widely used in a range of model organisms, positional cloning of causal variants is still a very laborious process. Here, we describe a novel universal approach, named fast forward genetics that combines traditional bulk segregant techniques with next-generation sequencing technology and targeted genomic enrichment, to dramatically improve the process of mapping and cloning multiple mutants in a single experiment. In a two-step procedure the mutation is first roughly mapped by ‘light’ sequencing of the bulk segregant pool, followed by genomic enrichment and deep-sequencing of the mutant pool for the linked genomic region. The latter step allows for simultaneous fine-mapping and mutation discovery. We successfully applied this approach to three Arabidopsis mutants, but the method can in principle be applied to any model organism of interest and is largely independent of the genome size. Moreover, we show that both steps can be performed in multiplex using barcoded samples, thereby increasing efficiency enormously. Inducible overexpression of the RETINOBLASTOMA-RELATED (RBR-OE) gene in Arabidopsis roots causes the complete differentiation of stem cells and premature differentiation of daughter cells, leading to a full exhaustion of the primary root meristem. In order to identify regulators of RBR function in cell differentiation, RBR-OE plants in the Columbia background (Col0) were treated with EMS mutagenesis and a set of genetic suppressors of RBR-OE, which restores root growth capacity, were isolated. In this study, we used one the identified suppressor lines, which segregated as a recessive mutation. Mapping populations were generated by outcrossing to Ler ecotype. Seedlings from the F2 population were grown for 15 days post germination (dpg). A pool of 60 seedlings each with a clear suppressor phenotype (homozygous for suppressor mutation) and of 60 seedlings showing RBOE phenotype (Heterozygous for the suppressor mutation) were prepared and genomic DNA was isolated with the RNeasy Plant Mini Kit from QIAGEN according to manufacturer's protocol. The other two, mutants 136 and 193 were obtained in fluorescence based mutant screen and a QCmarker based mutagenesis, respectively. Mutants were generated by chemical mutagenesis (EMS) in Colombia (Col) genetic background. Mutants were subsequently crossed to the Landsberg (Ler) ecotype to create the mapping populations. Bulk-segregant pools of about 200 mutant as well as wild-type plants were generated for every mutant line.
Project description:We identified a 14-3-3 homolog (maf1) in an EST library made under conditions conducive to aflatoxin biosynthesis. Disruption of this gene in A. flavus by site directed mutagenesis abolished aflatoxin production. The maf1 mutant was morphologically similar to wild type but had reduced conidiation and growth on some media. DNA expression analysis of the wild type and maf1 mutant revealed that the mutation affected the expression profile of a set of genes associated with aflatoxin production. Keywords = Aspergillus Keywords = aflatoxin Keywords = 14-3-3
Project description:To further investigating the novel NGS-defined HCC-associated SNVs in small S region involve hepatocarcinogenesis pathways, the plasmid pEGFP-N1-HBVS-genoB was used as the template for mutagenesis to create mutations T216C, A273G, and double mutant T216C/A273G at small surface (S) region, with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid pEGFP-N1-HBVS-genoC was used as the template for mutagenesis to create mutations A293G, C446G, A456G, A293G/C446G, C446G/A456G, A293G/A456G, and triple mutant A293G/C446G/A456G at small S region. Wild-type or mutant envelope expression vectors were transfected into Huh7 cells by using lipofectamine 3000 (Thermo Fisher Scientific) according to the manual of the manufacturer. All mutants were confirmed by DNA sequencing. all the novel NGS-defined HCC-associated SNVs in small S region could involve hepatocarcinogenesis pathways with inducing cell apoptosis, regulating cell death and survival, affecting DNA replication, recombination, and repair.