CwlD has sequence similarities to N-acetyl muramoyl-L-alanine amidases, a class of enzymes known to cleave the bond between the peptide side chain and the N-acetyl muramic acid residue in cortex peptidoglycan formation during sporulation. A major difference between vegetative peptidoglycan and spore peptidoglycan is the presence of muramic-<FONT FACE="Symbol">d</FONT> -lactam (MAL) in spore peptidoglycan. It was previously determined that a <I>cwlD</I> null mutant does not contain muramic-<FONT FACE="Symbol">d</FONT> -lactam in the spore cortex peptidoglycan and the mutant spores were unable to complete germination. Therefore, it is believed that CwlD plays a role in MAL formation during sporulation. However, the specific role of the protein had not been demonstrated. It was also previously found that <I>cwlD</I> is in a two-gene operon with <I>orf1</I>. Orf1 is produced within the forespore with CwlD. The hypothesized role of Orf1 is to inhibit CwlD activity from within the forespore. Muramoyl-L-alanine amidase activity was demonstrated by CwlD <I>in vivo</I>. Therefore, CwlD is carrying out the first step of MAL synthesis, cleaving the peptide side chain while other enzymes are needed to complete MAL formation. Two different forms of CwlD were over-expressed, with and without the protein's signal peptide sequence. Both forms of the protein were purified and in both cases activity was undetectable. Antibodies specific for CwlD were obtained which can be used in future research as a tool to further characterize CwlD activity. A series of <I>B. subtilis</I> <I>cwlD</I> operon mutants were constructed altering the expression patterns of Orf1 and CwlD within the mother cell and forespore compartments. Various resistance properties and the germination ability of the mutant dormant spores were analyzed. It was determined that the absence of just Orf1 or Orf1 and CwlD from within the forespore has no effect on the phenotypes tested. Peptidoglycan from developing mutant forespores was extracted and analyzed throughout sporulation. Evidence was obtained demonstrating that the role of Orf1 is not to inhibit CwlD from within the forespore as hypothesized. / Master of Science

Call number: LD2668 .T4 1984 L33 / Master of Science

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Master of Science / Graduate Biochemistry Group / Michael R. Kanost / Prophenoloxidase-activating proteinase (proPAP) activates prophenoloxidase when bacteria or fungi invade Manduca sexta. Upon activation, phenoloxidase initiates synthesis of melaninin, which can encapsulate the invaders and kill them. M. sexta contains three proteases that can activate prophenoloxidase, proPAP1, proPAP2, and proPAP3. The study of proPAP function has been slowed by the difficulty of expressing the proteins in recombinant systems. ProPAP1 contains one clip domain and one serine proteinase domain, a simpler structure than proPAP2 and proPAP3, which have two clip domains. For this reason, proPAP1 was selected for this investigation, to develop an improved system for expression of recombinant proPAP zymogens. In past experiments proPAP1 had a low expression level in insect cells using a baculovirus vector. In Escherichia coli, proPAP1 was expressed as an insoluble protein that could not be refolded successfully. The Bacillus subtilis expression system offers a potential improvement for expression of recombinant clip domain proteases because it can secrete recombinant proteins into the medium, it is a Biosafety Level 1 organism that is easy to handle, and it is less expensive to culture than insect cells. Four constructs for expression of proPAP1 and proPAP1 mutants were produced in the plasmid shuttle vector pHT43, which is compatible with both E. coli and B. subtilis. Experiments were carried out to test and optimize expression and purification of proPAP1 in B. subtilis. Conditions were optimized for IPTG (isopropyl β-D-1-thiogalactopyranoside) concentration, IPTG induction time, growth medium and induction temperature. Results showed that 0.5mM IPTG with 20 hours induction at 37°C in 2xYT medium was the optimum condition for proPAP1 production in the B. subtilis system. The recombinant proPAP1 was precipitated from the medium in 50% saturated ammonium sulfate and partially purified by nickel affinity chromatography. In addition to the full length proPAP1 protein, degradation of proPAP1 was also observed. Further experiments should be done to try to solve this problem. With purified protein, future work can be aimed at study of the structure and function of proPAP1.

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Thesis (Ph. D.)--University of Chicago, Department of Biochemistry and Molecular Biology, 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

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