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Subject: search.filters.subject.Klebsiella oxytoca

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    Analysis of gate residues in the type 2 secretin PulD : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Biochemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2012) Whitaker, Rowan
    Secretins are gated outer-membrane channels with large internal pore sizes (6-10 nm). They are the outer membrane components of bacterial trans-envelope complexes that assemble/export filamentous bacteriophages as well as pili, complex protein toxins and virulence factors. 12-14 identical subunits form the radially symmetrical channels which share a common architecture - a 3-tiered barrel with middle septum. Secretins are essential components of Gram-negative Type 2/3 secretion systems, spanning the outer membrane and interacting with the inner membrane components of transport machinery. Since secretins have such large pore diameters a simple channel would allow noxious compounds through the normally impermeable outer membrane. The presence of a gate structure allows for the controlled opening and closing of secretin channels, in response to specific cues regulating protein export. Here I have determined gate-structural elements of the Klebsiella oxytoca Type 2 Secretin, PulD. Random mutagenesis coupled with selection for open or 'leaky'-gate phenotypes created a library of mutations which were mapped by DNA sequence analysis. Analysis of leaky mutants revealed 12 distinct missense point mutations in pulD. Additionally, two deletion mutants were isolated, spanning 5 and 9 amino acids, both conferring a leaky gate phenotype. Comparison of these pulD mutations with those previously identified in another secretin gene encoding the Escherichia coli filamentous phage f1 secretin pIV, reveals mutations in both are localised in two main clusters that correspond to regions within the secretin homology domain. Named GATE1 and GATE2, these clusters indicate functional gate regions in both secretins.
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    Controlling biofilm development on ultrafiltration and reverse osmosis membranes used in dairy plants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatu, New Zealand
    (Massey University, 2011) Tang, Xuemei; Tang, Xuemei
    This study aimed to develop improved cleaning strategies for controlling biofilms on the surfaces of membranes used in dairy ultrafiltration (UF) and reverse osmosis (RO) plants. Eleven UF / RO membrane modules from 7 different New Zealand dairy membrane processing plants were received after typical cleaning-in-place (CIP) procedures. Microorganisms were isolated from both the retentate and permeate sides of these membrane surfaces and from the liquids collected from a UF membrane plant. Also some foulants scraped from a RO membrane were tested. The routine CIP currently used in the dairy plants was not adequate to completely remove organic material, including microbial cells, proteins and carbohydrates from the membrane surfaces. These residues may influence the surface characteristics and interactions between microorganisms and membranes and thus affect biofilm formation. Thirteen isolates including both bacteria and yeast were identified using biochemical techniques. Klebsiella oxytoca were isolated from 3 different membrane plant sites. This is, so far as we know, the first report of K. oxytoca being isolated from dairy membrane surfaces. The ability of the 13 strains to attach to negatively charged polystyrene surfaces was tested using a microtitre plate assay. Three K. oxytoca strains demonstrated higher ability to adhere than the other strains, suggesting that these strains might play an important role in developing biofilms on dairy membrane surfaces. Two K. oxytoca strains (K. B006 from plant A, UF and K. TR002 from plant C, RO) that performed best in the microtitre screening assay with respect to attachment capabilities were chosen for the remainder of the study. The cell surface hydrophobicity of all isolates was determined using the microbial adhesion to hydrocarbon assay (MATH) and the cell surface charge was determined by measuring the surface zeta potential. These two characteristics did not show a clear relationship with the adherence of the isolated strains. However, it was found that bacterial attachment was enhanced in the presence of whey or mixed strains. A commercial biofilm reactor CBR 90 was modified for developing biofilms on membranes and investigating strategies for biofilm removal. Biofilms of single and dual K. oxytoca strains were developed under a continuous flow of whey. The saturated biofilm was approximately 8 log10 CFU cm-2. The results of our study suggested that the whey protein concentration, membrane type including membrane material (polyethersulfone (PES) and polyvinylidene fluoride (PVDF)), membrane age (used and new), bacterial strain and the interactions between different microorganisms are all significant factors for biofilm development on membrane surfaces. Three enzymatic cleaners and four sanitisers, including sodium hypochlorite (pH 6.5, 200 ppm free available chlorine (FAC)), Perform® (peracetic acid/hydrogen peroxide, 2% v/v), ozonated water (pH 7.0, 0.5 ppm free available ozone (FAO)) and anolyte of MIOX® electrolysed water (EW) (pH 6.8, 120 ppm FAC) were tested for their efficacies in killing culturable cells from biofilms formed by single or dual K. oxytoca strains on used PES membrane surfaces. With no sanitation applied, two of three enzymatic cleaners performed better than sodium hypochlorite (pH 10.8-11, 200 ppm FAC) commonly used for CIP of UF membranes in the dairy industry. The four sanitisers were used to treat the membranes after a CIP wash regime. The results indicated that if a dairy processor were to use a standard CIP on membrane systems, then a further flush with MIOX® EW anolyte would reduce residual attached microbial populations further. In addition, using protease followed by a sanitation (sodium hypochlorite, Perform® or anolyte of MIOX® EW) produced the best clean based on a greater than 2 log reduction in residual cells and left no culturable and viable cells at a detection limit of 0.1 log10 CFU cm-2.