Massey Documents by Type

Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294

Browse

Subject: search.filters.subject.Molecular microbiology

Search Results

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Evolution of the spherical cell shape in bacteria : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany, New Zealand
    (Massey University, 2019) Yulo, Paul Richard Jesena
    Cell shape is an important feature of bacterial cells. It is involved in critical aspects of bacterial cell biology such as motility, growth, and the evasion of predators. Despite this, how cell shape has evolved in bacteria is unclear. For most rod-shaped bacteria, the maintenance of cell shape depends primarily on the bacterial actin-like protein, MreB. In this study, we show that the deletion of MreB from the rod-shaped model organism Pseudomonas fluorescens SBW25 results in the formation of aberrant spherical cells that have increased size and reduced fitness. This new MreB-null strain (ΔmreB) is susceptible to mechanical damage and grows poorly due to cell division defects. Furthermore, synthesized peptidoglycan (PG) chains were shorter and cell wall assembly was disorganised in this strain. A 1,000-generation evolution experiment comprised of multiple independent lineages produced spherical cells that have a reduced cell size and improved fitness. Mutations in the PG synthesis protein PBP1a were found across multiple lineages. Genetic reconstructions demonstrated that these mutations have a loss-of-function effect that reduced PG cross-linking and restored the ordered assembly of the cell wall, thereby reducing cell size and improving fitness in MreB-null cells. In one lineage, a five-gene deletion that included the gene coding for the outer membrane channel OprD was found to be beneficial. This deletion reduced cell size, improved fitness, and restored orderly cell wall construction. The mechanism responsible for this is unknown, but it may be related to modifications in septum localisation via the Min system. Finally, we show using phylogenetic analysis that PBP loss is a general trend in bacteria that evolved to become spherical, hinting at a plausible strategy for the evolution of the spherical cell shape from rod-shaped progenitors.
  • Thumbnail Image
    Item
    Identification of an immunogenic 18 kDa protein of Helicobacter pylori using alkaline phosphatase gene fusions : a thesis presented in partial fulfillment of the requirement for the Doctor of Philosophy in Molecular Microbiology, Massey University, Palmerston North, NZ
    (Massey University, 1999) Oliaro, Jane
    Secreted or surface-associated proteins play an important role in the immunopathogenesis of Helicobacter pylori infection. The aim of this study was to identify, using a genetic approach, H. pylori exported proteins and assess their role in the host immune response to infection. As part of this work, an H. pylori expression library was constructed and screened with a monoclonal antibody raised to a component of outer membrane vesicles from H. pylori, identified and characterised in a separate study. The screening strategy identified a locus of the genome containing two genes encoding exported proteins. Subsequent expression studies identified the gene product detected by the antibody as Lpp20, which encodes a well characterised lipoprotein from H. pylori. In addition, the use of alkaline phosphatase (AP) gene fusion methodology enabled the identification of a large number of other H. pylori exported proteins. Immunoscreening of a selection of enzymatically active H. pylori AP fusion proteins was carried out by Western blot analysis with patient sera and lymphocyte proliferation assays using peripheral blood mononuclear cells from H. pylori infected individuals. These assays identified a novel H. pylori exported antigen which was recognised by antibody derived from H. pylori infected individuals. Southern blot analysis revealed that the gene encoding the protein was absent in other Helicobacter species tested and sequence analysis of the gene demonstrated that it is highly conserved among H. pylori isolates. In order to obtain pure recombinant protein, the gene encoding the protein was cloned and expressed as a Beta-galactosidase (β-gal) fusion in Escherichia coli and the protein purified by affinity chromatography. The size of the recombinant protein released (18 kDa) was consistent with the calculated molecular mass of the polypeptide deduced from the DNA sequence. In Western blot assays, the purified protein was recognised by 71% of sera taken from patients infected with H. pylori, but by only 16% of sera taken from patients with unrelated or with no gastrointestinal disease. These results indicated that the 18 kDa protein from H. pylori was immunogenic and expressed in vivo. In other experiments, it was found that oral administration of this antigen did not protect mice against H. pylori colonisation following challenge with H. pylori.
  • Thumbnail Image
    Item
    A study of the intestinal microbiota in health and disease : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2005) Stewart, Jessica Anne
    The intestinal microbiota is a massive and complex community, essential to the human host for good health and well-being. However, this population has been associated with gastrointestinal disease, and remains poorly understood. The aim of this study was to develop and validate DNA-based assays for the intestinal microbiota and to apply these methodologies to faecal samples collected from healthy volunteers and patients with gastrointestinal disease. Over 250 faecal samples were analysed using temporal temperature gradient gel electrophoresis (TTGE) and real time PCR. Validated assays had high sensitivity and reproducibility. Healthy individuals displayed a high level of temporal stability during short term studies (≤ 6 weeks) and long term studies (1-4years). Analysis of faecal samples provided by identical and fraternal twins demonstrated an influence of host genetics over the composition of the predominant bacteria in children. Two intervention studies, bowel lavage and the Atkins' diet, were carried out to monitor the impact of environmental change on the population's stability in healthy volunteers. Following bowel lavage, microbial populations rapidly recovered to control densities, however the stability of the population was disturbed. Introduction of the Atkins' diet, led to a significant change in the composition of the microbial population. A preliminary study of the intestinal microbiota in disease groups was undertaken. Significant differences were detected between inflammatory bowel disease groups and controls. Cluster analysis in these patients indicated a potential association between the composition of the predominant bacterial population and disease localisation. The studies reported here demonstrate that the faecal microbiota in healthy individuals is a highly stable population under the influence of both host genetics and environmental variables, however the population present in patients with inflammatory bowel disease exhibits differences compared to healthy controls.