Browsing by Author "Palmer, Jon Stuart"

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    The multiple proteolytic enzymes of two microsporum species : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1995) Palmer, Jon Stuart
    Dermatophyte infections can be contracted from animals, humans or from the soil. In the genus Microsporum some species commonly are associated with cats & dogs but also often cause infections in humans. Others are regarded as non-pathogenic & are commonly isolated from the soil. The present studies investigated the production of proteolytic enzymes by the zoophilic species M.canis & the geophilic species M.cookei, in various cultural conditions which might affect expression of such enzymes, in an attempt to detect differences between the two that could be associated with the ability of M.canis to invade skin in vivo. Biochemical assays showed M.canis produced higher azocollytic & elastase activity in a keratin containing medium(BSW) than in Sabourauds Broth(SDB). In contrast, azocollytic & elastase activity of M.cookei in the two media was relatively similar. Azocollytic & elastase activity of both species peaked in the pH range 7-10 & azocollytic activity demonstrated highest activity around 45°C in both media. Both species produced some keratinolytic activity in BSW but not in SDB. Inhibition studies of azocollytic & elastase activity revealed the presence of an aspartic elastase with little or no azocollytic activity, which also was not detected using a substrate(gelatin) SDS-PAGE technique. Other proteinase types found were serine, cysteine & metalloproteinases. Using the gelatin-SDS-PAGE technique, the mode of culture(shake & stationary) & the effect of substrate, time & temperature were analysed to compare the effects these factors may have on proteolytic enzyme expression between the two species.Substrate proved to be the most important factor in the expression of gelatinases. Mode of culture in SDB demonstrated that some proteinases were expressed in shake culture sooner than in stationary cultures. M.canis in both SDB & BSW produced 6 bands between 85,000 Da & 13,000 Da. M.cookei in SDB produced 7 bands between 64,000 Da to 19,000 Da but in BSW only 5 bands between 61,500 Da to 19,000 Da. Inhibition studies revealed that both species expressed several metalloproteinases & serine proteinases in BSW which were not expressed in SDB cultures. It is suggested that these proteinases may be important factors in the ability of dermatophytes to colonise keratin & possibly, in the case of M.canis, to invade skin in vivo.
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    Surface characteristics of an adhesive thermophilic spore-forming Bacillus, isolated from milk powder : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2008) Palmer, Jon Stuart
    The growth of thermophiles during the manufacture of milk powder leads to a progressive increase in the number of thermophilic bacteria contaminating the final product. The limited residence time of the milk in the plant during milk powder manufacture and the concentration effect of converting milk into milk powder cannot explain the number of thermophiles found in the final product. This suggests that thermophiles are attaching to the large surface area of stainless steel found within a milk powder plant and then growing and developing into biofilms, with individual cells and/or biofilm fragments sloughing off into the product line and thus contaminating the final product. The aim of the present study was to investigate the attachment mechanisms that enable the thermophile Anoxybacillus flavithermus (B 1 2) to attach to stainless steel surfaces. Passing a B 1 2 culture through a column of stainless steel chips, collecting the first cells to pass through, re-culturing and repeating the process six times, resulted in the isolation of a mutant, labelled X7, with lO-fold reduced ability to attach to stainless steel as well as a reduced ability to attach to plastic and glass. A comparison of bacterial cell surface properties indicated that X7 was less hydrophobic than its parental strain B 1 2 . Cell surface charge measurements also suggest that X7 has less net negative surface charge. Disruption of extracellular polysaccharides and DNA appeared to have no effect on the attachment process. Removal of surface proteins caused a reduction in attachment of B 1 2 and X7 as well as a reduction in surface hydrophobicity suggesting surface protein involvement in both. Analysis by two-dimensional gel electrophoresis of lysozyme/mutanolysin extracted surface proteins revealed two proteins expressed at reduced levels in X 7 compared with B 1 2 . One protein was identified by mass spectrometry as the cytoplasmic enzyme Formate acetyltransferase. The role of Formate acetyltransferase and the second unidentified protein on the attachment process of Anoxybacillus flavithermus remains unclear.