Protein phosphorylation is utilized universally as a mechanism of signal transduction. However, the use of tyrosine phosphorylation by bacteria has been a matter of dispute. Conventional wisdom dictated that "prokaryotic phosphorylation" was typified by phosphorylation of histidine and aspartate residues of proteins, while "eukaryotic phosphorylation" was characterized by modification of serine, threonine, or tyrosine residues. Increasing numbers of reports have emerged challenging the traditional view of "prokaryotic" and "eukaryotic" phosphorlyation. One of the strongest links unifying prokaryotic and eukaryotic protein phosphorylation to date is IphP, a genomically-encoded dual-specificity protein phosphatase from the cyanobacterium Nostoc commune UTEX 584 bearing the active-site signature sequence of eukaryotic tyrosine-specific and dual-specificity protein phosphatases. The catalytic properties and substrate specificity of IphP were examined in detail. The enzyme was able to discriminate among a variety of exogenous peptides and proteins. Kinetic studies revealed that IphP favors protein / peptide substrates over low molecular weight compounds. Heparin effected IphP activity in a substrate-dependent manner. Enzyme activity toward casein (P-Ser) and MAP kinase (P-Thr/P-Tyr) was stimulated in the presence of the polyanion, whereas activity was inhibited by heparin toward other protein substrates. Both stimulation and inhibition by heparin were dose-dependent. The ability to stimulate IphP activity toward select substrates was attributed to the ability of heparin to recruit the enzyme and substrate to the same microenvironment. To facilitate future genetic studies examining the role of tyrosine phosphorylation in cyanobacteria, we searched for evidence of protein tyrosine phosphorylation in Anabaena PCC 7120. In a collaborative effort with the laboratory of Dr. Potts, tyrosine phosphorylated proteins were identified in Anabaena utilizing several approaches, including comparative labelling with alpha- vs gamma-32P-ATP, phosphoamino acid analysis, and selective hydrolysis with a tyrosine specific protein phosphatase. Together, these data unequivocally demonstrate the presence of tyrosine-phosphorylated proteins in Anabaena PCC 7120. Extracts of Anabaena PCC 7120 were examined for protein tyrosine phosphatase activity. An apparent PTP activity was detected, partially purified, and characterized. The protein phosphatase was ~38kDa by SDS-PAGE and sucrose density gradient centrifugation and displayed dual-specificity protein phosphatase (DSP) activity in vitro. The enzyme was localized to the periplasm and was thus assigned the title PAD, for Periplasmic Anabaena DSP. Periplasmic phosphoproteins of ~120 and 55 kDa that had been radiolabelled in vitro were dephosphorylated by partially purified PAD. PAD activity varied in vivo ~5-fold in a rhthymic, seemingly diurnal manner. Periplasmic proteins, including the 55kDa protein, were labelled in vivo and the degree of radiolabel incorporated into these proteins varied inversely with PAD activity. / Ph. D.

Cyanobacteria of the Nostoc genus are known for the thick, mucilaginous carbohydrate coatings that they produce. In this work, two examples of cyanobacterial glycobiology are considered, each of which involves a cyanobacterium of the Nostoc genus. The first portion of this work details attempts to obtain amino acid sequence information from the enzymes (glycosyltransferases) that are responsible for producing the extracellular polysaccharide (EPS) of Nostoc commune DRH1, ultimately to allow the transfer of this capacity to another organism. Two artificial substrates were synthesized for use in a capillary electrophoresis-based enzyme assay, which was used to look for glycosyltransferase activity in Nostoc commune DRH1 cell extracts. Glucuronosyltransferase activity was detected in association with Nostoc commune membrane material. The active enzyme displayed a divalent cation metal dependence (Mg+2) that is typical of glycosyltransferase enzymes purified from other organisms. Because the enzyme responsible for this activity held the potential to be EPS-related, its purification was attempted. The capillary electrophoresis-based enzyme assay and a 32P-labeled affinity tag were utilized to follow the glucuronosyltransferase enzyme through successive purification steps. The active enzyme was extracted from Nostoc commune membrane material using Triton X-100, and then purified by anion exchange chromatography. The active detergent extract was extremely unstable, and consequently, other purification techniques tested were unsuccessful in enriching activity. Affinity-labeling experiments indicated that the active enzyme was forming protein aggregates during these procedures, which were not amenable to in-gel protease digestion and peptide analysis by tandem mass spectrometry. The second portion of this work describes an investigation of an Anabaena (Nostoc) PCC 7120 soluble cell extract. Upon separation by sodium dodecyl sulfate ¡V polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent periodic acid-Schiff (PAS) staining of the resulting gel, the components of this cellular fraction produce a ladder-like pattern, which suggests that the extract may contain glycosylated protein. Analyses of several samples that were taken from within the PAS-staining region of such a gel revealed surface layer homology (SLH) domain-containing proteins, likely candidates to be covalently attached to or non-covalently interacting with carbohydrate. Various protein sequence analyses indicated that the detected SLH domain containing proteins belong to a family of (putative) cyanobacterial porins. Proteins in this family possess features that include a N-terminal signal sequence, a single SLH domain motif, followed by a coiled-coil region, and a C-terminal region that is homologous to the b-barrel-forming region of bacterial porins. All of these features were identified in the detected Anabaena (Nostoc) PCC 7120 SLH domain-containing proteins. Smith degradation was performed on a sample that was electroeluted from the PAS-staining region of a preparative-scale SDS-PAGE gel of the soluble cell extract. Subsequent analyses of the resulting sample by SDS-PAGE and mass spectrometry indicated that at least two SLH domain-containing proteins, encoded by all4499 and alr4550, were non-covalently interacting with the PAS-staining material. Following degradation, the PAS-staining material was still of sufficient size to detected by gel electrophoresis, and it continued to migrate in the absence of an interacting protein component. Protease digestion of a similarly prepared sample, and then subsequent analysis by SDS-PAGE and mass spectrometry, revealed that the region between amino acid residues #504 and #536, in the protein encoded by the alr4550 open reading frame, was interacting with the PAS-staining material. Monosaccharide composition analyses of this material revealed more carbohydrate constituents than are found in cyanobacterial primary (peptidoglycan) cell wall polymer alone, indicating that it contained a significant secondary cell wall polymer component as well. / Ph. D.

Bibliography: leaves 121-139. The aim of this study was to determine the extent to which protein synthesis inhibition, lowered glutathione (GSH) levels and toxin metabolism contribute to the toxicity of cyclindrospermopsin. Both hepatocyte cultures and reticulocyte lysates were utilized as in vitro tools of investigation. The findings imply that the inhibition of protein synthesis by direct action of the toxin cannot be considered a primary cause of hepatocyte cell death over an acute time frame. Cytochrome P450-derived metabolites may play a crucial role in cytotoxicity, and the toxicity process does not appear to involve oxidative damage.

published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

No description available.

No description available.

Due to the recent persistence of Listeria monocytogenes in food factory environments and an increase in outbreaks of Listeriosis, in particular some associated with duck meat products, an investigation was carried out to establish the potential effects of different growth conditions on the hydrophobicity of L. monocytogenes cells and to determine the behaviour of the cells in a minimal nutrient environment. It was found that duck meat extracts increased growth rate but did not alter the surface charge of the cells and when grown in minimal 010 and MCDB202 media the cells flocculated and showed more hydrophobicity than when grown in the rich media BHI. The modified surface of the organism behaved like an emulsifier and this led to the discovery for the first time, that there was formation of capsular exopolymeric substances (EPS) on the surface of planktonic cells of L. monocytogenes. After confirmation of the capsular EPS by two capsule stains, namely Nigrosin and Giemsa, the EPS was purified and proved to play a role in holding the cells together. It was also found to absorb water rapidly and can retain water for long periods suggesting that the EPS can contribute to the desiccation tolerance of L. monocytogenes cells embedded in a biofilm matrix. Chemical characterization of the EPS showed high levels of glycerol and phosphate indicating that the EPS is amphiphatic and may contain mainly glycerolphosphates.

Listeria monocytogenes is a common food borne pathogen which is an important contaminant found in various food factory environments, because of its ability to survive in a wide range of environmental conditions, and to grow at refrigeration temperatures. L. monocytogenes has caused both occasional outbreaks and sporadic cases of food-borne illness characterised by high mortality rates. In the UK and other European countries, there has been a conspicuous rise in the number of reported cases of “Listeriosis” recently. Hence, development of efficient and rapid methods for detection of this microorganism in various foods is of great significance for the food industry; and is needed to ensure the safety of foods that are considered at high risk of contamination. Conventional bacteriological methods (e.g. ISO 11290-1/A1) for the detection and quantification of L. monocytogenes are laborious and time consuming. Therefore, development of a rapid and reliable test capable of detecting very low numbers of the organism in ready-to-eat products is required. To address this, a phage amplification assay has been developed as a rapid method for the detection of L. monocytogenes using the broad host range phage A511. Successful development of the assay required identification of a virucide that could achieve inactivation of the phage without affecting the viability of the target cell to be detected. Several different substances were evaluated as potential virucides, and among the tested materials, tea infusions were found to be the most effective virucidal agent for this experiment. The efficacy of the new assay was tested using Stilton cheese, as a representative high risk dairy product, and a method was developed to use centrifugation to concentrate bacterial cells present in samples of half-Fraser broth enrichments. The cells were detected by using the new phage amplification assay and this combination of techniques was shown to be able to detect low numbers of cells in shorter times than can be achieved using conventional culture methods. An additional molecular identification step was also developed so that the identity of the cells detected could be confirmed using a multiplex PCR which targeted conserved regions of the Listeria 16S rDNA genes. In this assay, two amplified DNA fragments were generated confirming the presence of Listeria genus (400 bp band) and also L. monocytogenes species (287 bp band). An advantage of this combined phage-PCR method its ability to detect only viable cells in food samples. The combined assay was then tested on a wide range of spiked food samples, including Camembert cheese, pasteurised milk, minced meat, turkey meat and smoked salmon. The obtained results showed that the limit of detection was as low as 20 (± 5) cfu per 25 g, and duration needed for the detection and molecular conformation of speciation was 2 days (44 h), compared to 5 days using conventional culture methods. The combined phage-PCR assay was able to achieve a sensitive and specific identification of viable L. monocytogenes present in foods within 48 h, and therefore would allow for rapid screening of food products prior to release from the factory.

The synthetic polymers reported in this thesis are able to bind the small molecule autoinducer-2 (AI-2) in the Quorum Sense (QS) pathways of the marine organism with high affinity, and some of the polymers are also able to sequester rapidly the same bacteria from suspension. Specifically, the Alizarin Red S (AR-S) assay was used to compare binding interactions of boric and boronic acid with diol species, and interactions were further probed by 11B-NMR spectroscopy and Mass spectrometry. Dopamine was considered as a potential AI-2 scavenger for polymeric QS control owing to the high binding affinities for boron. Therefore, poly{N-(3,4-dihydroxyphenethyl) methacrylamide-co-N-[3-(dimethylamino)propyl] methacrylamide} [p(DMAm-c-DMAPMAm)] and poly(3,4-dihydroxy-L-phenylalanine methacrylamide) [p(L-DMAm)] were prepared via Reversible Addition Fragmentation Chain Transfer (RAFT) polymerization and characterized by 1H-NMR spectroscopy. The activities of these catechol polymers and carbohydrate-based poly(β-D-glucosyloxyethyl methacrylate) (p(GlcEMA)) in QS interference was demonstrated by bioluminescence assays with the Vibrio harveyi MM32 strain and by bacterial aggregation experiments. Polymersomes were then investigated as artificial protocells, with a view to establishing polymer vesicle containers as both reservoirs of QS mediated molecules, and of binding QS agents and bacteria. Hydrophobic monomers N-(2-Ethylhexyl) acrylamide [p(2-EHAm)] and N-phenylacrylamide [p(PAm)] were therefore polymerized into block copolymers from p(L-DMAm)-RAFT agents. The membrane permeability of polymersomes was measured via encapsulation and release of dyes, while the morphologies were examined with Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). Polymersomes were also investigated for potency in QS quenching via the bioluminescence assay and bacterial aggregation experiments. Initial studies of a communication feedback loop between bacteria and polymersome-encapsulated QS agents were performed again via bioluminescence assays. The results reveal that the investigated polymersomes exhibit potent activities in QS quenching, and further development might act as components of a synthetic biology approach to combating microbial pathogenicity.