-
1رسالة جامعية
المؤلفون: Allcock, David
المساهمون: Ferguson, Gail., Sawyer, Lindsay
مصطلحات موضوعية: 571.29, cell envelope polysaccharides, growth conditions, glucan, capsular polysaccharide, lipopolysaccharide
الوصف: Photobacterium profundum SS9 is a γ-proteobacterium which grows optimally at 15°C and 28 MPa (a psychrophilic piezophile) and can grow over a range of temperatures (2-20oC) and pressures (0.1-90 MPa). Previous research had demonstrated that P. profundum SS9 adapts its membrane proteins and phospholipids in response to growth conditions. In this study, methodology was developed for growing P. profundum SS9 under cold temperatures and high pressures in both liquid and solid cultures. The effect of changing growth conditions on cell envelope polysaccharides was then investigated. The lipopolysaccharide (LPS) profile of a rifampicin resistant P. profundum SS9 derivative, SS9R, was shown to change at 0.1 MPa with respect to temperature and at 15°C with respect to pressure. Compositional analysis showed that the LPS was almost entirely composed of glucose. This provides evidence that, under these conditions, the major polysaccharide produced by P. profundum SS9 is a glucan. Two putative polysaccharide mutants, FL26 & FL9, were previously isolated from a screen for cold-sensitive mutants of P. profundum SS9R. Both mutants displayed an increased sensitivity to cold temperatures on solid medium and were unaffected in their growth at high pressure. FL26 was found to exhibit an LPS alteration similar to previously published O-antigen ligase mutants, providing evidence that this mutant is likely to lack O-antigen ligase. Interestingly, FL26 was also shown to have a reduced ability to form biofilms and had increased swimming motility. This suggests that there are a number of changes which occur in FL26 in the absence of O-antigen. FL9 was found to have an altered LPS and capsular polysaccharide (CPS), similar to an E. coli wzc mutant. In E. coli, Wzc is involved in the polymerisation and transport of CPS, disruption of which can also lead to LPS alterations. The LPS and CPS alterations may lead to the cold-sensitivity phenotype, either individually or in combination. In conclusion, alterations in the cell envelope polysaccharides were shown to affect cold temperature sensitivity on solid agar. Cold-sensitivity is most likely directly related to the LPS alterations and stability of the membrane under cold temperatures. Exopolysaccharides (EPS) have previously been shown to affect desiccation and freezethaw resistance, making it is possible that the CPS plays a similar role in this case.
-
2رسالة جامعية
المؤلفون: Whale, Gary Anthony
المساهمون: Emmison, N., Morrison, A., Pretswell, E.
مصطلحات موضوعية: 612, Lipid macroamphiphiles, Hydrophobicity, Cell envelope polymers, Lipid metabolism
الوصف: Lipid macroamphiphiles (LMAPs) are cell envelope components of bacteria that have been extensively associated with the pathogenesis of disease. It has been reported that these components can also influence the in vivo metabolism of lipids in mammals. For the onset of the skin condition acne vulgaris, in addition to the presence of Propionibacterium acnes on the skin, changes in the composition of skin lipids has been identified as an important etiological factor in the development of the disease. To date, the LMAP of P. acnes has not been purified and, for this reason, the significance of these components remains unclear. In order to study their significance, purification of the LMAP was essential for resolving their structures and diverse biological effects. Following the extraction and consequent purification of lipoteichoic acid (LTA) from S. aureus by hydrophobic interaction chromatography (HIC), a similar method was applied to an extract from P. acnes. The resulting amphiphilic fractions were analysed using SDS-PAGE and found to contain two discrete components. To facilitate the further characterisation, these components were separated on a larger scale by preparative SDS-PAGE and purified from the gel via electro-elution. The purified components were designated LMAP1 (running in the 24-29kDa region) and LMAP2 (running in the 14-20kDa region). Analysis of the LMAP species suggested a lipoglycan structure and revealed evidence for a lipid anchor based on phosphatidylinositol with fatty acids that were comparable to the whole cell fatty acid profile. The polysaccharide moiety of both LMAP1 and LMAP2 contained different relative amounts of mannose, glucose and galactose, and it was these differences that affected the size and hydrophobicity of the two lipoglycan components. Additionally, an amino sugar was detected that was suspected of being a diaminohexuronic acid. This component has been previously identified as a component of the P. acnes cell wall polysaccharide. Consequently, it is proposed that a relationship might exist between these distinctive cell envelope polymers. The effects on lipid metabolism were investigated in an in vitro system using the combined lipoglycan from P. acnes and compared to both LTA and lipopolysaccharide (LPS). All LMAP components investigated inhibited the uptake of fatty acid by hepatocytes. Furthermore, secretion of lipid from hepatocytes was also inhibited following incubation with each of the LMAP components. However, LTA had only a marginal effect in comparison to LPS and lipoglycan. It was also shown that both LPS and lipoglycan had a direct effect on adipocytes by increasing the uptake of fatty acid. Additionally, lipid secretion within these cells was also stimulated, which propounded an increase in fatty acid secretion. This evidence suggested that the lipoglycan of P. acnes can significantly influence the metabolism of lipids by mammalian cells and therefore these lipoglycans might play a crucial role in the pathogenecity of P. acnes and thus in the development of disease.
-
3رسالة جامعية
المؤلفون: Zupanc, Marianne Michaela
مصطلحات موضوعية: 579, Bacterial cell envelope
الوصف: The maltose binding protein (MalE) of Escherichia coli is a secreted 370 residue polypeptide which serves as the periplasmic receptor for high-affinity membrane transport of maltose and maltodextrins. Cells tolerate synthesis and translocation across the plasma membrane of large quantities of MalE. Similarly, the outer membrane protein Omp A is not toxic when expressed in physiological amounts. Expression of an ompA-malE hybrid gene consisting of the first 274 residues of OmpA and the last 251 residues of MalE was, however, toxic, although synthesis of the OmpA part alone was not. To examine the effect of synthesis of the MalE part of the hybrid, the malE fragment was cloned into a vector where it was preceded by the gene coding for the OmpA signal peptide and was inducible by isopropy Ithiogalactoside. Upon induction, a polypeptide of the expected size was made, but expression of the truncated MalE turned out to be even more toxic than that of the OmpA-MalE hybrid. The MalE fragment was exported to the periplasm and did not interfere with the export of other secreted polypeptides. A search for suppressors of this toxicity was performed. In a chromosomal gene bank of E. coli, such a suppressor was found which coded for a 102 A-terminal residue fragment of the 217 residue protein NlpE (new outer membrane lipoprotein). NlpE is known to combat the toxicity of the cytosolic ß-galactosidase (LacZ) when artificially exported to the periplasm. This suppression is achieved by activation of the Cpx two-component signal transduction pathway controlling expression of the periplasmic protease DegP. Increased synthesis of DegP caused degradation of LacZ. The same mechanism appears to operate for the degradation of the MalE fragment. In a degP background, the NlpE fragment could not suppress toxicity. Mutants (cpxA*) exist which, without any signal, produced increased levels of DegP. In a cpxA* strain, the MalE fragment was no longer toxic. The toxicity caused by the MalE fragment can still not entirely be explained. However, several lines of evidence, such as the expression of the periplasmic spheroplast protein Y in cells producing MalE251, suggest that the most likely explanation for the lethality is a defect in the cell wall of induced cells.
