Graduation date: 1995

Graduation date: 1993

Protein synthesis is a highly conserved process across all domains of life, both structurally and functionally. This cyclic process is catalyzed by numerous soluble protein factors that interact with the ribosome to facilitate efficient protein synthesis. Many canonical translation factors bind and hydrolyze GTP to induce conformational changes that facilitate translation. For example, GTP hydrolysis by EF-Tu is required for the release of aminoacyl-tRNA into the ribosomal A site; GTP hydrolysis by EF-G facilitates the movement of tRNA and mRNA from the A site to the P site of the ribosome. However, protein synthesis seems to also have a requirement for ATP; the essential yeast protein eEF-3 facilitates release of deacyl-tRNA from the ribosomal E site. In Escherichia coli, the protein product of the open reading frame yhih has been suggested to have a similar function. However, the role of this unique prokaryotic protein is not understood. Preliminary characterization of this protein suggests a nucleotide-dependent conformational change occurs in a truncated form of the protein, ΔP541 Yhih. Interestingly, this phenomenon is not observed in ΔL432 Yhih. Both ΔP541 Yhih, and to a lesser extent ΔL432 Yhih, exhibit a ribosome-dependent ATPase activity, suggesting the primary region for binding with the ribosome lies between Leu432 and Pro541. / x, 101 leaves : ill. ; 29 cm.

Shiga toxin-producing E. coli (STEC) including E. coli O157:H7 are potential food and water borne zoonotic bacterial pathogens capable of causing outbreaks of severe illness in humans. The virulence of E. coli O157:H7 strains may be related to the type of Stx produced and several Stx2 variants have been identified which appear to differ in their ability to cause disease. Two lineages exist within O157 strains where lineage I is associated mainly with human and bovine isolates and lineage II is associated mainly with bovine isolates. The goal of this study was to identify and characterize a lineage II EC970520 Stx2c phage and determine if variations in the phage compared to Stx2 phage found within the lineage I strain, EDL933, can result in differences in virulence observed between the lineages. This study suggests: 1) that the lineage II strain EC970520 contains a highly heterogeneous Stx2c variant phage; 2) that location of integration of the phage within the genome of a bacterium may be important for host selection; 3) that EC970520 Stx2c phage genes are lineage II specific but only a subset of EDL933 phage genes are lineage I specific; 4) that differences in the stability of phages within bacteria contribute to the evolution of new pathogens; 5) that variation in phage genes can be used to detect different strains of E. coli O157:H7 and other STEC; and 6)that the type of phage may result in phenotypic differences between lineages and occurrence of human disease. Results of this study indicate that lineage II strains may be less virulent than lineage I strains due to specific genetic differences and the ability to release phage which is important to the evolution of new pathogenic strains. / xv, 162 leaves : ill. ; 29 cm.

Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology)in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has been a valuable tool in creating enzyme variants that are capable of withstanding the extreme environments of industrial processes. Xylanases are a family of hemicellulolytic enzymes that are used in the biobleaching of pulp. Using directed evolution, a thermostable and alkaline stabl xylanase variant (S340) was created from the thermophilic fungus, Thermomyces lanuginosus. However, a host that was capable of rapid growth and high-level expression of the enzyme in large amounts was required. The insert containing the xylanase gene was cloned into a series a pET vectors in Escherichia coli BL21 (DE3) pLysS and trimmed from 786 bp to 692 bp to remove excess fungal DNA upstream and downstream of the open reading frame (ORF). The gene was then re-inserted back into the pET vectors. Using optimized growth conditions and lactose induction, a 14.9% increase in xylanase activity from 784.3 nkat/ml to 921.8 nkat/ml was recorded in one of the clones. The increase in expression was most probably due to the removal of fungal DNA between the vector promoter and the start codon. The distribution of the xylanase in the extracellular, periplasmic and cytoplasmic fractions was 17.3%, 51.3% and 31.4%, respectively. The modified enzyme was then purified to electrophoretic homogeneity using affinity chromatography. The xylanase had optimal activity at pH 5.5 and 70°C. After 120 min at 90°C and pH 10, S340 still displayed 39% residual activity. This enzyme is therefore well suited for its application in the pulp and paper industry.

Errata sheet pasted opposite Table of Contents. / Includes bibliographies. / 142, [171] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis investigates the UV-induced inhibition of oriC initiation function and the nature of the phenotype of the mutant origin of replication of Escherichia coli, oriCc. The specific aims of this study are to delineate the source of the UV-induced trans-acting inhibition of oriC function, utilizing the phage vector [lambda]poriCc, and to demonstrate the oriCc allele as an enhanced DNA replication initiator from oriC. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1997?

Bacterial species and communities play foundational roles in human health and therapeutics, in vital ecological and environmental processes, and in industrial applications for the biosynthesis of valuable compounds and materials. However, existing genetic engineering methods and technologies available for bacterial functional genetics or large-scale genomic integration are inefficient, unable to translate between different target species, or lacking precise targeting or reprogramming capabilities. In this work, we describe a novel class of CRISPR- associated transposons (CRISPR-Tn) that facilitate programmable RNA-guided DNA insertions. In particular, the Tn6677 CRISPR-Tn system from Vibrio cholerae comprises a Tn7-like transposase machinery that has co-opted a nuclease-deficient Type I-F3 CRISPR-Cas system to guide its target selection. We show that, similar to canonical CRISPR-Cas systems, this CRISPR- Tn system can be easily programmed using the CRISPR RNA (crRNA) spacer sequence, and directs highly target-specific DNA integration into the Escherichia coli genome. After defining their core biological and mechanistic principles, we developed these CRISPR-Tn systems into a genome engineering platform, which we named INTEGRATE (Insertions of Transposable Elements by Guide RNA-Assisted Targeting). Particularly, optimization of V. cholerae Tn6677 (Vch INTEGRATE, or VchINT) produced a system capable of programmable, broad-bacterial- host, and multiplexed integration of DNA payloads up to 10 kilobases in length, with genomic editing efficiencies reaching 100%. Our single-plasmid expression of system components enabled, for the first time, genome engineering of specific target strains within a complex fecal bacterial community. In addition, we performed extensive deep sequencing within transposition experiments to characterize and examine non-conventional transposition products, including cointegrates formed through replicative transposition, and long-range integration events resulting from on-target DNA binding. Finally, by individually inserting transposon ends into the E. coli genome, we demonstrated successful transposition-mediated mobilization of a genomic fragment 100 kilobases (kb) in length, demonstrating engineering at the genome-scale using VchINT. Altogether, this work highlights the potential of VchINT and other CRISPR-Tn systems as next- generation genome engineering technologies in bacteria and beyond.

Biofilm formation may increase survival and persistence of Escherichia coli in the highly variable conditions of soil environments, though it remains unknown the extent variation in biofilm formation affects survival. We asked what genetic traits influence biofilm formation in phylogroup D E. coli isolates from surface soils, and are they associated with the soil environment? Biofilm density was analyzed and compared with soil environment characteristics. Isolates produced more biofilm per unit growth at 15°C than 37°C. Biofilm formation was greater in soil isolates than fecal isolates and in soils with moisture and higher calcium and pH levels. A GWAS analysis found variants involved in cell envelope formation and structure were associated with biofilm formed at 37°C, and stress response and iron acquisition variants were associated with biofilm formed at 15°C. Motility variants were associated with a negative effect on biofilm formed and adhesion variants associated with a positive effect. / National Science Foundation (NSF) award no. DEB-1453397 to P.W.B. / ND-EPSCoR

The regulation of short chain fatty acid metabolism has been examined. Metabolism of acetoacetate, and short chain fatty acids such as butyrate and valerate, is predicated upon the expression of genes of the ato operon. Acetoacetate induces expression of a CoA transferase (encoded by the atoDA genes) and expression of a thiolase (encoded by the atoB gene). Metabolism of saturated short chain fatty acids requires the activities of the transferase and thiolase and enzymes of 6-oxidation as well. Spontaneous mutant strains were isolated that were either constitutive or that were inducible by valerate or butyrate instead of acetoacetate.

No apparent repression of pyr gene expression in Pseudomonas aeruginosa is observed upon addition of exogenous pyrimidines to the growth medium. Upon introduction of the subcloned Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) into a P. aeruginosa pyrB mutant strain, repression was observed in response to exogenously fed pyrimidine compounds. The results proved that it is possible to bring about changes in pyrimidine nucleotide pool levels and changes in transcriptional regulation of gene expression as a result. Thus, the lack of regulatory control in P. aeruginosa pyr gene expression is not due to an inability to take up and incorporate pyrimidine compounds into metabolic pools, or to an inability of the RNA polymerase to respond to regulatory sequences in the DNA but is probably due to a lack of specific regulatory signals in the promoter of the genes themselves.