Browsing by Subject "transposon mutagenesis"
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Item Enhanced Hydrogen Production in Escherichia coli Through Chemical Mutagenesis, Gene Deletion, and Transposon Mutagenesis(2011-08-08) Garzon Sanabria, Andrea JulianaWe demonstrate that hydrogen production can be increased by random mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and that hydrogen production can be further increased in the chemically-mutagenized strain by targeted gene deletion and overexpression of genes related to formate metabolism. Chemical mutagenesis of Escherichia coli BW25113 hyaB hybC hycE::kan/pBS(Kan)-HycE to form strain 3/86 resulted in 109 +/- 0.5- fold more hydrogen; 3/86 lacks functional hydrogen uptake hydrogenases 1 and 2, has hydrogenproducing hydrogenase 3 inactivated from the chromosome, and has constitutively active hydrogenase 3 based on expression of the large subunit of hydrogenase 3 from a high copy number plasmid. Deleting fdoG, which encodes formate dehydrogenase O, (that diverts formate from hydrogen), from chemical mutagen 3/86 increased hydrogen production 188 +/- 0.50-fold (relative to the unmutagenized strain), and deletion of hycA, which encodes the repressor of formate hydrogen lyase (FHL), increased hydrogen production 232 +/- 0.50-fold. Deleting both fdoG and hycA increased hydrogen production 257 +/- 0.50-fold, and overexpressing fhlA along with the fdoG hycA mutations increased hydrogen 308 +/- 0.52-fold. Whole-transcriptome analysis of chemical mutagen 3/86 revealed 89 genes were induced and 31 genes were repressed. In an effort to identify chromosomal mutations in chemical mutagen 3/86, we performed comparative genome sequencing and identified two chromosomal loci with mutations in coding regions of ftnA and yebJ; however, neither gene was related to the increased hydrogen production as determined by the close vial (short) hydrogen assay. In addition, transposon mutagenesis, which is one of the most efficient strategies for creating random mutations in the genomic DNA, was performed in two different strains: E. coli BW25113 hyaB hybC hycA fdoG::kan/pCA24N-FhlA and E. coli MG1655 to identify beneficial mutations for hydrogen production. As a result of screening 461 E. coli BW25113 hyaB hybC hycA fdoG::kan/pCA24N-FhlA transformants and 1000 E. coli MG1655 transformants, three interesting mutations have been discovered in E. coli BW25113 hyaB hybC hycA fdoG::kan/pCA24N-FhlA transformants (gpsA, dipZ, glgP) and 1 beneficial mutation in E. coli MG1655 transformants (malT). When any of these genes gpsA, dipZ, or glgP is disrupted by Tn5 insertion, hydrogen production decreases 17, 3 and 8-fold, respectively. Additionally, when malT gene is disrupted by Tn5 insertion, hydrogen increases 3.4-fold.Item Identifying Coxiella burnetii Genes Essential for Subversion of the Host Immune System(2014-11-13) Rowin, Kristina DCoxiella burnetii, the causative agent of Q fever in humans, is a Gram-negative intracellular bacterium. Although the organism was first isolated in the 1930s, little is known about the specific mechanisms underlying its virulence. This is largely due to its obligate intracellular lifestyle. Recent advances in both axenic growth and genetic manipulation of C. burnetii allowed efficient generation and isolation of random mutations and enabled more definitive studies of the genes essential for virulence. The goal of this project was to generate a large collection of specific isogenic C. burnetii mutants and employ in vitro and in vivo screens to determine the individual contributions of their affected genes to pathogenicity. We used a Himar1 transposon system to generate a library of 1) defined clonal mutants and 2) pools of random transposon mutants in order to approach saturation with non-lethal mutants of the low virulence C. burnetii isolate, Nine Mile, phase II (NMII), RSA 439, which is approved for use in a biosafety level two laboratory. Mutants from both libraries were compared in various growth conditions or infection models to identify differences in growth phenotype relative to wild-type C. burnetii. The libraries are also amenable to high-throughput analysis using transposon sequencing or transposon directed insertion site sequencing (TraDIS) to compare pooled mutants between input and output infection assays. In this study, we optimized methods to generate defined transposon mutants, resulting in mutations in nearly 20% of the predicted open reading frames (ORFs) and provide methodology to expand the library for future studies. Included in these mutants were a number of bioinformatically predicted virulence factors based on phenotypes in other bacterial pathogens that we further compared in cellular and animal models of infection. Our findings are consistent with previous studies that demonstrate the Dot/Icm T4BSS is essential for generating and replicating in a large parasitophorous vacuole (PV). We developed methods for high throughput screening of Tn mutants in vitro by imaging on either confocal microscope or BioTek Cytation3 imaging system. We generated a genome saturation transposon mutant pool by combining transposon mutant pools from 35 independent transformation reactions. These combined in vitro and in vivo screens dramatically improve our knowledge of specific virulence determinants for this pathogen and provide a substantial amount of data for future studies.