Title page for ETD etd-04082010-155812


Type of Document Dissertation
Author Heffron, Jared David
Author's Email Address heffronj@vt.edu
URN etd-04082010-155812
Title The Roles of the Germination-Specific Lytic Enzymes CwlJ1, CwlJ2, and SleB in Bacillus anthracis Spores
Degree PhD
Department Biology
Advisory Committee
Advisor Name Title
Popham, David L. Committee Chair
Larson, Timothy J. Committee Member
Melville, Stephen B. Committee Member
Sumner, Susan S. Committee Member
Keywords
  • cortex
  • lytic enzyme
  • anthrax
  • Bacillus anthracis
  • spore
  • germination
Date of Defense 2010-03-26
Availability restricted
Abstract
The Bacillus anthracis spore is highly resistant to environmental stresses, but cannot cause anthrax until it successfully germinates. An essential step of germination, degradation of the cortex peptidoglycan layer, is carried out by germination-specific lytic enzymes (GSLEs). While the GSLEs of several other Bacillus species have been investigated, they have not been characterized in the pathogen B. anthracis. In this work three GSLEs, CwlJ1, CwlJ2, and SleB are identified in B. anthracis and are investigated in order to better understand their functions.

Genetic manipulation of cwlJ1, cwlJ2, and sleB was fundamental to this work. First, reporter gene fusions revealed that all three are expressed during spore formation and that CwlJ1 is likely the most abundant GSLE in the spore. Second, gene deletions eliminating each GSLE enabled the observation of mutant phenotypes during spore cortex degradation. CwlJ1 and SleB were identified as the most critical GSLEs for successful germination. High-performance liquid chromatography and mass spectroscopy revealed that SleB is required for lytic transglycosylase activity, but CwlJ1’s mode of action was unclear. Multiple mutations of all of the GSLEs revealed that CwlJ2 is the least active GSLE, but that it participates in germination in response to Ca-Dipicolinic acid; a role it shares with the more dominant CwlJ1.

Purification of the CwlJ1 and SleB proteins permitted in vitro assays of enzymatic activity as measured by changes in substrate optical density, solubility, and product formation. While CwlJ1 was recalcitrant to these methods, it was observed to cause cortex hydrolysis independently. SleB was more amenable and it was discovered to contain a peptidoglycan-binding domain that is primarily responsible for substrate binding, and a lytic transglycosylase domain that facilitates cortex-specific hydrolysis by recognizing muramic-δ-lactam.

Future research will include determining the structure of SleB through x-ray crystallography and the identification of CwlJ1 activity by refining the protein purification method. The results of this and future research into CwlJ1, CwlJ2, and SleB may lead to a means to initiate spore germination prior to host infection. This will greatly ease spore decontamination measures, lower risk of infection, and discourage the use of B. anthracis spores as a biological weapon.

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