Helix hand inversion exhibited by Bacillus subtilis macrofibers is induced by changes in culture medium composition. The kinetics of this inversion are compared to those of temperature-induced inversions. D-alanine evokes a similar inversion process. The role of left-twist proteins(s), the existence of "memory", and the asymmetry of left to right versus right to left kinetics are confirmed within the context of these inversion regimes. Initiation time of right to left inversions is correlated to degree of pre-shift twist. Evidence is presented suggesting effective twist of the wall is defined by (1) the average of that twist conformation inserted prior to a shift in culture conditions and that of wall inserted following the shift and (2) the location of left-handed material within the wall. A constant 50 minute delay is observed before initiation of left to right inversions, irregardless of twist. Evidence is presented for a protein in the left to right inversion process. A classification system of macrofiber phenotypes based upon hand and degree of structural organization has been established. Three major classes are identified. Subclasses are shown to be distinguishable. Isotwist phenotypes of seven strains are defined upon a matrix of temperature and medium composition. These plots reveal a fundamental pattern of hand and organization that is present in each of the strains studied. The polarity of the four axes, the range of attainable twist conformations, and the existence of a right-hand maximum in the 12.5% SPl domain remain virtually constant. Major variations include extent of a disorganized band and/or the shifting of conformational range either left or right. Several mutants were transformed into A734, a strain that produces the tightest structures at all four matrix corners. Multiple mutations are responsible for the phenotypes of several strains. Evidence is presented for single genes that express as extreme left-handedness and stress at high temperature, swelling and stress in TB at high temperature, and reduction in structural organization produced in high TB content at low temperature.

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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.

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Protein-carbohydrate recognition is an important target for inhibitor development. Improved inhibitor design requires a fundamental molecular basis of these interactions. This thesis describes the preliminary structural studies on three carbohydrate processing enzymes, UDP-galactopyranose mutase, alpha-D-glucose-1-phosphate thymidylyltransferase and TDP-glucose 4,6-dehydratase. These enzymes are found in important human pathogens such as Mycobacterium tuberculosis and Salmonella typhimurium. The major focus of the thesis has been on UDP-galactopyranose mutase, the enzyme responsible for catalysing synthesis of the thermodynamically unfavourable 5 membered ring form of galactose, UDP-galactofuranose from the thermodynamically favoured 6 membered ring form, UDP-galactopyranose. UDP-galactofuranose plays a key role in mycobacterial cell walls. This thesis also describes work with concanavalin A. This legume lectin is an invaluable model for the study of protein-carbohydrate interactions. Two concanavalin A complexes are discussed. Both structures clear up misunderstandings in the literature and provide an insight into designing enzyme inhibitors.

Cells of S. marina (N.C.M.B, 778) were disrupted using a Hughes press and a purified cell wall fraction obtained using a previously reported method for halococcal wall isolation. This procedure was monitored by examination of thin sections in the electron microscope and the final wall preparation was seen to be relatively free of cytoplasmic and membranous contaminants. However, treatment of the wall fraction with crude trypsin did appear to remove particulate surface components. The total ninhydrin-positive components detectable accounted, for only about 14% of the cell wall dry weight. The major amino acids present were glycine, alanine, glutamic acid and aspartic acid although very small amounts of others were detected. The amino sugar components included glucosamine and galactosamine although these only accounted for some 60% of the total amino sugars. The remainder was probably made up of one or more of four unidentified, acid-labile components detected on amino acid analysis and by paper chromatography. This is in accord with the finding of unusual, labile amino sugars in the cell walls of other halococcal species. Approximately 37% of the cell wall dry weight was made up of the neutral sugars, glucose, galactose and mannose which were present in eguimolar amounts. In addition, the wall was found to contain a negligible lipid (0.1% dry weight) and a high ash (9.2% dry weight) content. The poor recovery of organic material after analysis is almost certainly due to the lability of some of the more unusual (and in this work unidentified) components. Attempts to selectively solubilise the wall material with a view to identifying discrete polymers met with some success. In Particular, treatment with trichloroacetic acid (TCA) at 35° extracted all of the glactosamine from the wall (in addition to other components) but none of the unknown component, X1. Further treatment with TCA at 60 extracted all of another unknown component, X1. These results suggest that some degree of resolution of different polymers constituting the wall may be possible and may have been achieved here. Treatment of S. marina with the antibiotics, D-cycloserine, novobiocin, bacitracin, penicillin G and vancomycin, known to affect cell wall biosynthesis in other bacteria, was carried out. Possible effects of the antibiotics were monitored by electron microscopy and turbidimetric estimation of bacterial growth. Only novobiocin and bacitracin had any effect on growth but this was marked; in both cases growth was prevented by addition of the antibiotic. The other three antibiotics all lost their antibiotic activity (against appropriate indicator organisms) when incubated over a period of a few hours in Dundas medium. It is suggested that this may be a significant consideration when explaining the antibiotic insensitivity of microorganisms, such as S. marina whose doubling times are of the same order of magnitude as that necessary for antibiotic inactivation. Thin sections of control and antibiotic-treated cells showed interesting ultrastructural features comparable with those seen in more conventional halophilic cocci. Some minor ultrastructural changes were seen in some of the anti-biotic-treated cells, the most notable being extensive plasmolysis in the case of novobiocin. However, none of the antibiotics tested appeared to cause cell lysis or osmotic fragility which may preclude their use as agents for the non-destructive removal of the cell wall.