We 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.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [48]-59).

Iron is extremely important for many organisms. Despite its abundancy, it exists in insoluble forms that makes its usability difficult. Some organisms secrete siderophores, low molecular weight compounds, that can chelate iron and convert it into usable forms for cells. One such organism, Rhizobium leguminosarum, is a nitrogen fixing symbiont proteobacteria that infects leguminous plants. The genome of Rhizobium leguminosarum ATCC 14479, which infects the red clover, Trifoli pratense, has previously been completely sequenced in our lab. Our lab has identified several genes in this strain involved in the biosynthesis of a siderophore, vicibactin. The protein product of one of those genes, VbsS, is hypothesized to be a non-ribosomal peptide synthase. It has been attempted to knockout the VbsS gene utilizing the ‘splicing by overlap extension’ method. Additionally, an in-silico analysis of the genome revealed the Vbs genes in R. leguminosarum ATCC 14479 strain were similar to genes in found in the proteobacterium Phyllobacterium sp. 628 and the fungi Aspergillus fumigatus Af293.

Chronic Granulomatous Disease is the most commonly encountered immunodeficiency involving the phagocyte, and is characterized by repeated infections with bacterial and fungal pathogens, as well as the formation of granulomas in tissue. The disease is the result of a disorder of the NADPH oxidase system, culminating in an inability of the phagocyte to generate superoxide, leading to the defective killing of pathogenic organisms. This can lead to infections with Staphylococcus aureus, Psedomonas species, Nocardia species, and fungi (such as Aspergillus species and Candida albicans). Involvement of vital or large organs can contribute to morbidity and/or mortality in the affected patients. Major advances have occurred in the diagnosis and treatment of this disease, with the potential for gene therapy or stem cell transplantation looming on the horizon. © 2011 Song et al; licensee BioMed Central Ltd.

Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish (ESC), which is the most economically important disease of farm-raised channel catfish. E. ictaluri is considered a facultative intracellular pathogen like other well-known species in the Enterobacteriaceae, and it is capable of surviving inside channel catfish neutrophils and macrophages. Its ability to survive inside neutrophils and macrophages has made the development of an effective vaccine against ESC particularly challenging. The goal is to develop a safe, efficacious live attenuated ESC vaccine that is practical and economically beneficial to catfish producers. In this study, single and combination of mutations in genes encoding TCA cycle enzyme and C-1 metabolism proteins were constructed using inrame mutagenesis. The virulence, vaccine efficacy, and tissue persistence of the constructed single and combination mutants were determined in channel catfish. The constructed mutants EideltasdhC, Eideltamdh, EideltafrdA, EideltaglyA, EideltasdhCdeltamdh, EideltasdhCdeltafrdA, and EideltasdhCdeltagcvP were significantly attenuated and showed 100% protection against E. ictaluri 93-146 infection in juvenile channel catfish. However, when tested in 15-d old catfish fry, mutant EideltasdhCdeltagcvP and EideltafrdA were found to provide good protection (99% and 60%, respectively) against E. ictaluri 93-146 infection. The tissue persistence study indicated higher tissue concentration in mutants EideltasdhCdeltagcvP and EideltafrdA relative to the tissue concentration in EideltasdhC and EideltasdhCdeltafrdA mutants.

C. difficile is an obligate anaerobic, Gram-positive, rodshaped and spore-forming bacterium. It is a well-recognised causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis. C. difficile has emerged as an important nosocomial pathogen in recent years, associated with considerable morbidity, mortality and economic burden. Despite its importance, functional genomic studies have been lagging behind in comparison to other enteric pathogens. This is attributed to the fact that C. difficile is difficult to manipulate genetically and the lack of robust, reproducible mutagenesis systems for many years. The ideal mutation for robust functional genomic studies is a markerless, in-frame deletion of the gene of interest. All systems developed for C. difficile, up to the start of this study, involve insertional inactivation of the gene of interest. This study describes the development of a novel genetic system for C. difficile, to create precise and markerless chromosomal deletions, using the meganuclease ISceI. For validation of the system, the addBA genes in C. difficile were deleted. The AddAB enzyme complex is important in the survival of many bacteria, since it maintains genome integrity, by the repair of double-strand breaks. Deletion of addBA in C. difficile did not significantly affect growth and viability, but the mutant strains were sensitive to DNA damaging agents. In addition, it was shown that C. difficile is capable of initiating the SOS response after DNA damage and that AddAB is not necessary for the induction of this response. The genetic system was further optimised to delete type IV pili (TFP)- associated genes, particularly pilT (CD3505) and pilA (CD3507), to investigate twitching motility. TFP are important in virulence and pathogenesis of many bacteria and twitching motility is often involved. TFP in C. difficile may be expressed in vivo during infection and may be involved in biofilm formation and colonization. To study potential TFP-mediated motility, a non-flagellated C. difficile strain was first constructed by deleting the fliC gene. The pilT gene, predicted to encode a protein involved in TFP retraction, was then deleted in the ΔfliC strain. A ΔpilT strain was also generated. Preliminary experimental work using these strains did not show any evidence for twitching motility and no difference between the ΔpilT strains and the parental strains. Examination of cells from the ΔfliC strain, under various conditions, did not reveal any pili, which indicates that TFP are regulated in C. difficile and that the TFP locus might be repressed at the transcriptional level. Preliminary work to investigate an intergenic region located upstream of the TFP locus in C. difficile, that might be involved in regulation, suggested that transcription is being initiated within a 500 bp region upstream of the CD3513 gene.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

We propose a new, continuous model of the fractionation process (duplicate gene deletion after polyploidization) on the real line. The aim is to infer how much DNA is deleted at a time, based on segment lengths for alternating deleted (invisible) and undeleted (visible) regions. After deriving a number of analytical results for "one-sided" fractionation, we undertake a series of simulations that help us identify the distribution of segment lengths as a gamma with shape and rate parameters evolving over time. This leads to an inference procedure based on observed length distributions for visible and invisible segments. We suggest extensions of this mathematical and simulation work to biologically realistic discrete models, including two-sided fractionation.