Student Theses and Dissertations

Date of Award

2010

Document Type

Thesis

RU Laboratory

Fischetti Laboratory

Keywords

Bacillus cereus, Bacillus anthracis, reticulocyte binding protein, ExsM protein, biofilm formation, Bacillus spore

Abstract

"Bacillus cereus is a Gram-positive spore-forming bacteria that can cause food poisoning and its close relative, Bacillus anthracis is the etiological agent of anthrax. In both cases, the spore, a differentiated cell type in a dormant state, starts the infection process. Thus, the exosporium, which constitutes the surface of the spore, plays an important role during natural infection of both B. cereus and B. anthracis. Proteins from the exosporium of B. cereus ATCC 4342, a B. anthracis-like strain, were extracted with 2% β-mercaptoethanol under alkaline conditions and identified by liquid chromatography coupled with tandem mass spectrometry. A novel cell surface protein, reticulocyte binding protein (Rbp), was identified in this sample. Inactivation of rbp by insertional mutagenesis resulted in spores devoid of the hair-like nap typical of the exosporium, which suggests that Rbp could be a new component of the exosporium nap or could have a role in its assembly. GerQ, a protein that is crosslinked by transglutaminase in the spore coat of Bacillus subtilis, was also identified in the B. cereus ATCC 4342 exosporium.Absence of GerQ during sporulation resulted in a brittle exosporium in both B. cereus ATCC 4342 and B. anthracis ΔSterne spores. This suggests that transglutaminase participates in exosporium assembly and maturation by crosslinking small proteins and processed peptides, providing structural stability and resistance to degradation to the spore surface. In addition, a novel exosporium protein, ExsM, was characterized in this study. Subcellular localization of an ExsM-GFP fusion protein revealed a dynamic pattern of fluorescence that follows the site of formation of the exosporium around the forespore. Under scanning electron microscopy, exsM null mutants presented a tightly wrapped exosporium resulting in smaller and rounder spores than wild-type spores that have an extended exosporium. Thin-section electron microscopy revealed that exsM spores were encased by a double layer exosporium, both of which were composed of a basal layer and a hair-like nap. Compared to wild-type spores, exsM spores were more resistant to lysozyme treatment, germinated with a higher efficiency, and had a delay in outgrowth. Insertional mutagenesis of exsM in B. anthracis ΔSterne rendered spores with a partial second exosporium that were also smaller in size. These findings suggest that ExsM plays a critical role in the formation of the exosporium. B.cereus, but not B. anthracis, spores possess long appendages projecting from their surface. B. cereus ATCC 14579 appendages were isolated from spores by extraction with 2% β-mercaptoethanol under alkaline conditions, followed by CsCl gradient ultracentrifugation. Mass spectrometry analysis revealed that camelysin (CalY) and spore coat-associated protein N (CotN) were associated with the appendages sample. Both proteins are homologous to TasA, the main component of B. subtilis biofim extracellular matrix. TasA forms amyloids fibrils that hold the cells together and provide structure to the extracellular matrix. Therefore, spore appendages may have a role in biofilm formation, acting like a scaffold for the biofilm matrix and holding the spores together."

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/369

Download

Included in

Life Sciences Commons

Share

COinS