Cell walls of Bacillus subtilis macrofibers have been biochemically analyzed to determine the contribution of various surface polymers in the twist regulation. Helix hand inversion was induced by a variation in either the growth temperature or the nutritional composition of the culture medium. Initial experiments had demonstrated a fivefold difference in the sensitivity of right- and left-handed forms to muramidases indicating modifications of peptidoglycan as a possible mechanism underlaying inversion. An examination of lysozyme susceptibility of purified cell walls and whole cells derived from the two structural forms, however, exhibited no significant difference suggesting loss of the relevant component(s), perhaps biomechanical in nature, during disintegration of macrofibers. The effect of various twist modulators such as trypsin, ammonium sulfate and D-alanine on the development of helical twist in both switchable and "fixed" mutants were studied. The interaction matrices have established D-alanine as the most potent of right-factors. Intestinal alkaline phosphatase is reported as a newly discovered antagonist to the development of leftward twist. Heat inactivation and protein purification experiments strongly indicated that twist modulation was due to the phosphatase activity rather than minor protease contaminants. The chemical composition of cell walls purified from right- and left-handed structures was determined. No twist correlated differences in the overall content of peptidoglycan, teichoic acid and teichuronic acid were detected. Evidence is presented for the absence of correlation between the extent of ester-linked alanine substitution and twist state. These findings suggest that gross changes in wall composition is perhaps not the mechanism for hand inversion. From the profiles of the wall associated proteins, a 200 Kdal band has been identified whose presence is strongly correlated with the development of leftward twist. This polypeptide was found to be highly sensitive to trypsin; a property it shares with a previously proposed left-twist protein. Preliminary evidence for isolation of left-hand specific polyclonal antibodies is also presented. FJ7, a switchable mutant, was successfully transformed with a plasmid containing the Streptococcus transposon Tn917. A small bank of insertional mutants has been constructed for the isolation of mutants impaired in helix hand inversion.

This thesis studies two particular bacterial outer membrane proteins called OmpC and Wzi, focusing on their expression, purification, crystallization and X-ray structure determination. A series of four naturally occurring OmpC mutants were isolated from a single patient with an E. coli infection of liver cysts. The isolated E. coli strains progressively exhibited increasing breadth of antibiotic resistance in which OmpC was predicted to take a partial role. We carried out an assay in which a strain of E. coli lacking OmpC was used to express the first (antibiotic sensitive) and the last (antibiotic resistant) of the clinical OmpC mutants and drug permeation assessed. Single channel conductance measurements were carried out and the X-ray structures for all the isolates were determined. Protein stability was assessed. With these data we propose that changes in the transverse electric field, not the pore size, underlie the clinically observed resistance to the antibiotics. This is the first demonstration of this strategy for antibiotic resistance. Wzi is a novel outer membrane protein involved in the biosynthesis and translocation mechanism of the K30 antigen from E. coli. The mechanism is a complicated process that requires several proteins including outer and inner membrane proteins. The protein Wzi was expressed, purified and crystallized. Initial crystals were tested and diffracted to 15Å. After optimization, a crystal diffracting to 2.4Å has been obtained.

Integral membrane enzymes contribute a unique repertoire to the cell, as they are capable of synthesizing products from substrates of different chemical character at the membrane-water interface. Membrane-embedded enzymes are often responsible for the synthesis of important components of the cellular membrane and contribute to the structural integrity of the cell, maintenance of cellular homeostasis and signal transduction. One of the main focuses of Dr. Filippo Mancia’s laboratory is understanding how enzymes complete these functions by investigating, at an atomic level, the determinants of substrate binding and catalysis within the membrane and at the membrane surface. Here I will present my investigation of two such integral membrane enzyme systems, which are responsible for the synthesis and processing of membrane-embedded molecules in bacteria. Phosphatidylinositol-phosphate Synthase (PIPS) Phosphaitylinositol (PI) is an essential lipid component in mycobacteria, demonstrated by loss of viability when PI is reduced to 50% of wild-type levels. Phosphatidylinositol (PI) is required for the biosynthesis of key components of the cell wall, such as the glycolipids phosphatidylinositol-mannosides, lipomannan and lipoarabinomannan. For these molecules, PI serves as a common lipid anchor to the membrane. In Mycobacterium tuberculosis, the disease causing pathogen of tuberculosis, these glycolipids function as important virulence factors and modulators of the host immune response. Therefore, the enzyme responsible for PI synthesis in this organism is a potential target for the development of anti-tuberculosis drugs. The defining step in phosphatidylinositol biosynthesis is catalyzed by a member of the CDP-alcohol phosphotransferase enzyme family. The enzyme uses CDP-diacylglycerol as the donor substrate, and either inositol in eukaryotes or inositol-phosphate in prokaryotes as the acceptor alcohol of the synthesis reaction. In prokaryotes, phosphatidylinositol-phosphate synthase (PIPS; a member of the CDP-alcohol phosphotransferase family) catalyzes this reaction to yield phosphatidylinositol-phosphate, which is then dephosphorylated to PI by an uncharacterized enzyme. Structures of PIPS from Renibacterium salmoninarum (RsPIPS), with and without bound CDP-diacylglycerol, have revealed the location of the acceptor site as well as molecular determinants of substrate specificity and catalysis of the enzyme. However, RsPIPS has low activity relative to PIPS from Mycobacterium tuberculosis (MtPIPS) and the two share only 40% protein sequence identity. Therefore, these initial structures have limited potential for meaningful homology modeling and drug design. Presented here are the structures of PIPS from Mycobacterium kansasii (MkPIPS), which is 86% identical to MtPIPS, in an apo state to 3.1 Å resolution, in a nucleotide-bound state to 3.5 Å resolution, and in a novel ligand-bound state to 2.6 Å resolution. This work provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis in the context of mycobacterial pathogens. RodA-PBP2 Complex The cell wall of most gram-negative and gram-positive bacteria (excluding atypical bacteria such as members of Mycoplasmataceae) is composed of peptidoglycan, a mesh of repeating carbohydrates (N-acetylmuramic acid, MurNAc, and N-acetylglucosamine, GlcNAc) cross-linked by small peptides. Peptidoglycan is essential for growth, division and viability of the organism. Any disruption of the biosynthesis of peptidoglycan, whether by genetic mutation, inhibition with antibiotics or degradation by lysozyme, results in bacterial cell lysis. Peptidoglycan helps maintain cell shape and serves as an anchor for accessory proteins and other cell wall components. As essential components of the cell wall, enzymes contributing to the peptidoglycan biosynthetic pathway can be exploited as antibiotic targets. After a hydrophilic peptidoglycan precursor (UDP-MurNAc-pentapeptide) is synthesized in the cytosol, it is attached to the lipid carrier undecaprenyl phosphate (UndP). The lipid-linked precursor (undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide or Lipid I) is modified further to undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-GlcNAc (Lipid II) by addition of a GlcNAc moiety. Lipid II is then flipped across the membrane to the periplasm where its sugars are polymerized to form the glycan strands of the peptidoglycan mesh. SEDS proteins, essential for maintaining bacterial processes that determine shape, elongation, cell division and sporulation, are integral membrane enzyme that have been implicated in this process as either Lipid II flippases, glycosyltransferases responsible for sugar polymerization, or both. SEDS proteins are also known to form a functional complex with type b penicillin-binding proteins (PBPs), which are known as transpeptidase enzymes, responsible for the crosslinking of peptides in the formation of the peptidoglycan mesh. Though structures of both RodA (a SEDS protein involved in bacterial growth and elongation) and type b PBPs are available, the interaction between the two proteins and their joint enzymatic activity is poorly characterized. Here, I present the preliminary structural characterization of a RodA-PBP2 protein complex by single-particle cryo-electron microscopy (cryo-EM). We hope this ongoing work will contribute to the understanding of these enzymes and to the development of antibiotics to combat antibiotic resistance.

Thesis (Ph. D.)--University of Alberta, 2009. / Title from pdf file main screen (viewed on Nov. 25, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Chemistry, University of Alberta." Includes bibliographical references.

x, 153, xxviii leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1975

v, 132 p. leaves : photos., graphs, tables ; 31 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--Dept. of Biochemistry, University of Adelaide, 1978

x, 153, xxviii leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1975

Thesis (Ph.D.) -- University of Adelaide, Dept. of Microbiology, 1975.

Thesis (Ph.D.)--Dept. of Biochemistry, University of Adelaide, 1978.

Thesis (M.S.)--University of North Carolina Wilmington, 2009. / Title from PDF title page (February 23, 2010) Includes bibliographical references (p. 55-61)