No description available.

Sinorhizobium meliloti forms a symbiosis with Medicago species of legumes. Within the legume root nodules, S. meliloti differentiates into a bacteroid, which fixes atmospheric nitrogen into ammonia for the legume. The legume produces hundreds of nodule-specific cysteine-rich (NCR) peptides, which mediate bacteroid differentiation. The S. meliloti BacA protein was the first bacterial factor identified to be essential for bacteroid development. BacA sensitises S. meliloti to certain antimicrobial peptides and influences the modification of the bacterial lipopolysaccharide (LPS) with a very-long-chain fatty acid (VLCFA). Therefore, it is thought that either the peptide uptake function or the role of BacA in LPS VLCFA decoration could be essential for survival of S. meliloti within the legume. In this PhD project, a role for BacA in the response of S. meliloti towards NCR peptides was investigated. It was determined that BacA protects S. meliloti from NCR-induced cell death. Furthermore, it was found that the structure and composition of the LPS plays a key role in the response of S. meliloti to NCR peptides. It was also shown that the peptide uptake function of BacA was conserved among different rhizobia. The role and biosynthesis of the LPS VLCFA in bacteroid development was also explored. It was determined that the acyltransferase but not the acyl-carrier-protein, was essential for the biosynthesis of VLCFA modified LPS in planta. Six genes, located in a gene cluster were proposed to be involved in the LPS VLCFA biosynthesis in rhizobia and my research found that this was the case. The outcome of this research has provided important insights into the mechanism of prolonged bacterial-host infections and the biosynthesis of unusual lipids.

Several isoforms of LPP have now been identified and cloned but remain to be purified. In the present study, a bacterial expression system was established and hexa- and deca-histidine epitope tagged-LPP1 and LPPla expressed in E. coli. In addition, a maltose binding protein (MBP) epitope tagged-LPP Ia was expressed in E. coli. Hexa- and deca-histidine LPP1 and LPPla, were partially purified using immobilised affinity chromatography. MBP-LPPla was expressed to higher levels than hexa- and deca-histidine LPP1 and LPPla in E. coli, most probably within insoluble inclusion bodies. In all cases, recovery of LPP activity was low. Membranes derived from HEK293 cells that stably over-express LPP I, LPP I a, LPP2 or LPP3 were used to demonstrate the differential hydrolysis of several molecular species of PA, LPA(18: 1), C8-CIP and SlP. Kinetic analysis using a multisubstrate assay system revealed that the LPP isoforms do not follow typical Michaelis-Menten kinetics towards most substrates under the assay conditions employed. The LPPs appear to show differential kinetics depending on the complement of substrates accessible to the enzymes. Stable over-expression of LPPI, LPPla, LPP2, but not LPP3, in HEK293 cells has previously been shown to attenuate the activation of ERK-1/2 by G-protein coupled receptors agonists such as SIP, LPA, PA and thrombin. The present study extended these observations by showing that basal growth rates were unaffected and that levels of mRNA transcript for the SIP, /EDGI receptor were reduced in the LPP stable cell lines but that this did not correlate with attenuation of the SIP-stimulated ERK-1/2 response. In addition, transient overexpression of LPPI, LPPIa and LPP2, but not LPP3 in HEK293 cells and GPASM cells also resulted in the attenuation of SIPinduced ERK-1/2 activation. Furthermore, transient transfection of a plasmid construct encoding the antisense sequence for LPP1 was also found to attenuate SIPinduced ERK-1/2 activation whereas the PMA-stimulated response was unaffected. Many questions remain to be fully answered in order to determine the physiological and pathophysiological. roles of the LPPs and the reason for the molecular diversity of the enzyme family.

Campylobacter is the commonest cause of human acute bacterial-enteritis in the developed world (ACMSF, 1993). Over the last ten years Great Britain has experienced an increase in the number of reported cases of campylobacter associated illness over the last ten years. There are numerous under reporting issues associated with campylobacter-related illness, and as such the actual number of cases that occur each year is unknown and the magnitude of the public health risk posed by this organism can only be hypothesised. Infection with campylobacter has been linked in epidemiological studies with the consumption of poultry, in particular chicken meat. A quantitative risk assessment (QRA) model has been produced to investigate this issue. Through the use of appropriate modelling techniques and collected data the QRA model assesses the risk of human infection with campylobacter consequent upon the consumption of a chicken meal. The model describes each of the stages of the chicken supply chain and the mechanisms by which the chicken/chicken product becomes contaminated was investigated thus allowing the identification of mitigation strategies, which can reduce such contamination. Model results estimate that the risk of infection with campylobacter associated with the consumption of a single serving of chicken has a mean value ranging from 0.040 to 0.070 with a 95th percentile ranging from 0.098 to 0.160. These results have been used as a benchmark to which the impact of mitigation strategies are compared. Results clearly show that a reduction in the national flock prevalence, combined with a reduction in the within flock prevalence of positive flocks can have a dramatic impact upon the risk of infection. Further, freezing of chicken meat prior to consumption also considerably reduces the estimates risk.

No description available.

The aims of this project were to develop a system for monitoring a continuous stream of high grade purified water for potential contamination by bacterial endotoxins. The monitoring system was to be designed so that it could be readily integrated within a closed water purification processing system. The project was viewed as a developmental stage towards the development of a commercial sensor with wide ranging applications within the pharmaceutical and environmental sectors. This text details the development of testing protocols for the examination of ultra pure water using different sensing matrices. The endotoxin structure is comprised of three main sections with specific chemistry. These regions have each been considered as potential areas for detection. The development of surface plasmon resonance (SPR) systems and protocols for the detection of endotoxin was shown both to be possible and practical within given experimental parameters. In order to assess the potential for this sensing within a more established experimental system and to further expand the potential sensing layers for endotoxins, further experiments were carried out using a BIAcore system. The use of the BIAcore allowed the examination of alternative sensing surfaces based on the specific nature of the endotoxin molecule rather than the use of literature based reactants that have previously displayed an affinity for the endotoxin molecules. The methods used within this project have concentrated on the overall chemistry of the endotoxin molecule. The potential binding/complexing agents have been targeted at the three principal regions of the endotoxin structure using the chemical nature of these regions as an attractive surface to the sensing layer.

Neisseria meningitidis is the most common cause of bacterial meningitis in the Western world and the second leading cause of mortality in 1-5 year-olds in the United Kingdom. A signature-tagged mutagenesis screen of approximately 3,000 insertional mutants of a serogroup B isolate of N. meningitidis, C311⁺, identified 73 genes required for pathogenesis in an infant rat model of meningococcal septicaemia. Homology-based searches indicate that two of the genes identified, NMB0342 and NMB0345, have homologues in other pathogenic bacteria and exist within a potential operon of seven genes (NMB0342-NMB0348). NBM0342 is a homologue of ispA (intracellular septation protein) of Shigella flexneri, required for effective intercellular spread and plaque formation in epithelial cells. NMB0345 is a homologue of cbf, a Campylobacter jejuni gene that encodes a 29 kDa protein which is a major antigenic peptide. PCR and Southern analyses of genes in the NMB0342-NMB0348 locus show that they are conserved across a wide range of pathogenic isolates and serogroups of N. meningitidis. However, these genes are present only in a subset of commensal strains. N. meningitidis mutants with transpoon insertions in NMB0342 and NMB0345 are highly attenuated when directly competed with the wild-type bacterium in the infant rat model of infection. Mutations have been introduced into other genes within the locus and the resulting mutants analyzed for their ability to cause systemic disease. Mutants with transpoon insertions in the NMB0343 and NMB0344 genes are significantly attenuated, while mutants with insertions in NMB0347 and NMB0348 are attenuated to a lesser degree. Complementation of the NMB0342 mutation by ectopic chromosomal integration of a wild-type copy of NMB0342 almost completely restores the virulence of the bacterium. In vitro cell-culture analyses and microscopic analysis of mutants in association with host cells demonstrate that a mutant with a transpoon insertion in NMB0345 is significantly reduced in its adherence to Chang epithelial cells compared to the wild-type bacterium. Whole blood and serum-sensitivity assays show that the NMB0342 and NMB0345 mutants are highly serum-sensitive compared to the wild-type bacterium. Preliminary experiments have demonstrated that immunization of mice with recombinant NMB0345 protein confers protection against challenge with the serogroup B isolate MC58. In vitro assays demonstrate that recombinant NMB0345 is an active peptidyl polyl cistrans isomerase (PPIase). Together, the results from these investigations support a role for genes of the NMB0342-NMB0348 locus in the pathogenesis of N. meningitidis infections. Further investigation is needed to assess the potential of NMB0345 as a vaccine candidate.

No description available.

It is the purpose of this thesis to report further studies on the vesicles appearing in phage lysates of Escherichia coli, phage attachment to these vesicles, and the presence of similar vesicles in lysozyme and penicillin lysed cultures.

The transport systems for proline and potassium represent two of the active transport systems in Escherichia coli. They have further similarities that their transport may be utilized as a response to osmotic perturbations in the environment. The exact mechanism of transport had not been totally elucidated. The transport of proline had been assumed to operate as a proton symport and as such had been used as a model system when other transport systems were being investigated. This study has demonstrated that the major route of proline uptake through the proline permease 1 (PP1), operates as a Na+ - proline cotransport which may accept Li+ in the place of Na+. Unusually, Na+ stimulates the Vmax of transport with little or no effect on the Km. In addition to this transport system, there are two other proline uptake systems which function primarily for the transport of betaine. The transport of K + is also facilitated by more than one system. The Kdp system is a K+ transporting ATPase; the TrkF system is a low rate transport system which may represent leak through another pathway. The TrkA transport system is the major system but the mechanism is not known. Transport through the system is energised by ATP and a pmf, while exchange through the system requires only ATP. The role of ATP was investigated in this study by the use of metabolic inhibitors and vesicles. It was determined that the availability of ATP affected the steady state level of potassium in the cells rather than the rate of potassium upake. It was speculated that ATP would act as a regulator of the system which would be driven by the pmf. ATP may regulate TrkA through phosphorylation or by allosteric modification of the carrier.